Thermodynamic Analysis of Interaction of eIF4E Protein with mRNA 5
Thermodynamic Analysis of Interaction of eIF4E Protein with mRNA 5
Thermodynamic Analysis of Interaction of eIF4E Protein with mRNA 5
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<strong>Thermodynamic</strong> <strong>Analysis</strong> <strong>of</strong> <strong>Interaction</strong> <strong>of</strong> <strong>eIF4E</strong> <strong>Protein</strong><br />
<strong>with</strong> <strong>mRNA</strong> 5' Cap Structure and 4E-BP1 or eIF4G<br />
<strong>Protein</strong> Fragments<br />
Anna NiedŸwiecka<br />
Praca doktorska<br />
pod kierunkiem dr. hab. Ryszarda Stolarskiego, pr<strong>of</strong>. UW<br />
Zak³ad Bi<strong>of</strong>izyki, Instytut Fizyki Doœwiadczalnej, Wydzia³ Fizyki<br />
Uniwersytet Warszawski<br />
Warszawa 2003
Recenzenci: pr<strong>of</strong>. dr hab. Kazimierz Wierzchowski<br />
dr hab. Maciej Geller<br />
II
Sk‡adam serdeczne podziŒkowania<br />
dr. hab. Edwardowi Dar¿ynkiewiczowi, pr<strong>of</strong>. UW, ktry swoim<br />
wysi‡kiem organizatorskim umo¿liwi‡ mi prowadzenie badaæ,<br />
oraz za za wsparcie w rozmaitych sytuacjach,<br />
dr. hab. Ryszardowi Stolarskiemu, pr<strong>of</strong>. UW, za wszechstronn„<br />
pomoc, cierpliwo i ¿yczliwe nastawienie do rozwijaj„cej siŒ<br />
tematyki pracy,<br />
pr<strong>of</strong>. dr. Stephenowi K. Burley’owi z Uniwersytetu Rockefellera<br />
w Nowym Jorku za inspiracjŒ,<br />
dr hab. Agnieszce Bzowskiej, dr Beacie Wielgus-Kutrowskiej,<br />
mgr Joannie fluberek, dr. hab. Janowi Antosiewiczowi, pr<strong>of</strong>.<br />
UW, p. dr hab. Ewie Kulikowskiej, p. Dorocie Haber oraz innym<br />
pracownikom Zak‡adu Bi<strong>of</strong>izyki za cenne dyskusje i<br />
zapewnienie mi‡ej atmosfery,<br />
dr. hab. Januszowi StŒpiæskiemu, dr Marzenie Jankowskiej-<br />
Anyszce i mgr Lidii Chlebickiej za syntezy chemiczne,<br />
biosyntezŒ bia‡ka oraz wiele po¿ytecznych wskazwek,<br />
mgr. Jzefowi Greupnerowi i mgr Urszuli Wonikowskiej-Bezak<br />
oraz lek. med. Tomaszowi Zawadzkiemu za skierowanie moich<br />
zainteresowaæ ku fizyce i biologii molekularnej,<br />
moim Rodzicom za wszelk„ pomoc.<br />
Praca by‡a finansowana przez Komitet Badaæ Naukowych (3 PO4A 071 22 - grant<br />
promotorski, 6 PO4A 055 17, 6 P04A 034 09), Human Frontier Science Program<br />
(RG0303/1998-M) i II. Amerykaæsko-Polski Fundusz im. Marii Sk‡odowskiej-Curie<br />
(MEN/NSF-98-337, MEN/NSF-96-257).<br />
III
Table <strong>of</strong> Contents<br />
ABBREVIATIONS ................................................................................................................VIII<br />
PREFACE................................................................................................................................XI<br />
1. INTRODUCTION ............................................................................................................. 1<br />
1.1. Brief Description <strong>of</strong> Biological Aspects................................................................................ 1<br />
1.1.1. Central Dogma <strong>of</strong> Molecular Biology............................................................................. 1<br />
1.1.2. <strong>Protein</strong> Functions.............................................................................................................. 2<br />
1.1.3. Translation Initiation........................................................................................................ 2<br />
1.1.4. Eukaryotic Initiation Factor 4E ....................................................................................... 5<br />
1.1.5. Translational Control ....................................................................................................... 9<br />
1.1.5.1. Regulation <strong>of</strong> eIF4F Complex Formation ................................................................ 10<br />
1.1.6. Clinical Aspects <strong>of</strong> the Cellular <strong>eIF4E</strong> Activity Control ............................................. 11<br />
1.1.6.1. Relation <strong>of</strong> <strong>eIF4E</strong> Activity to Immunology and Malignancy.................................. 11<br />
1.1.6.2. Relation <strong>of</strong> 4E-BP1 and eIF4G Activities to Human Health and Disease.............. 11<br />
1.1.6.2.1. Phosphorylation <strong>of</strong> 4E-BP1................................................................................. 11<br />
1.1.6.2.2. Dephosphorylation <strong>of</strong> 4E-BP1 and Cleavage <strong>of</strong> eIF4G..................................... 12<br />
1.1.6.3. Anticancer Gene Therapy and Pharmacotherapy..................................................... 12<br />
1.2. Some Remarks about <strong>Thermodynamic</strong> Approach to Molecular <strong>Interaction</strong>s in<br />
Solution................................................................................................................................................. 13<br />
1.2.1. <strong>Thermodynamic</strong> and Apparent Association Constant.................................................. 13<br />
1.2.2. Gibbs Free Energy.......................................................................................................... 14<br />
1.2.3. <strong>Thermodynamic</strong> Parameters <strong>of</strong> Macromolecular <strong>Interaction</strong>s in Solution.................. 15<br />
1.2.4. Noncovalent <strong>Interaction</strong>s in Aqueous Solutions........................................................... 17<br />
1.2.5. Parsing <strong>of</strong> Gibbs Free Energy........................................................................................ 18<br />
1.2.6. <strong>Thermodynamic</strong>s <strong>of</strong> Coupled Processes ....................................................................... 18<br />
2. AIM OF THE WORK...................................................................................................... 20<br />
3. MATERIALS AND METHODS ..................................................................................... 22<br />
3.1. Chemical and Biochemical Syntheses................................................................................. 22<br />
3.1.1. Cap Analogues ............................................................................................................... 22<br />
3.1.2. Peptides........................................................................................................................... 22<br />
3.1.3. <strong>Protein</strong> Biosynthesis....................................................................................................... 24<br />
3.2. Preparation <strong>of</strong> <strong>Protein</strong> and Peptide Samples to Spectroscopic Measurements ........... 24<br />
3.3. Absorption and Emission Spectroscopy............................................................................. 25<br />
3.3.1. Fluorescence Quenching................................................................................................ 26<br />
3.3.1.1. Static Quenching........................................................................................................ 26<br />
3.3.1.2. Dynamic Quenching.................................................................................................. 27<br />
3.3.1.3. Resonance Energy Transfer ...................................................................................... 27<br />
3.3.1.3.1. Resonance Energy Transfer in <strong>eIF4E</strong> ................................................................. 28<br />
3.3.2. Tryptophan Fluorescence in <strong>Protein</strong>s............................................................................ 28<br />
3.3.3. Quenching <strong>of</strong> <strong>eIF4E</strong> Fluorescence by Cap Analogues................................................. 29<br />
V
3.4. Experimental Conditions <strong>of</strong> Spectroscopic Measurements.............................................30<br />
3.4.1. Titration Assay................................................................................................................31<br />
3.4.2. Fluorescence Data Corrections ......................................................................................31<br />
3.4.2.1. Inner Filter Effect......................................................................................................31<br />
3.4.2.2. Dilution.......................................................................................................................32<br />
3.4.2.3. Fluorescence Drift in Time........................................................................................33<br />
3.5. Fluorescence Data <strong>Analysis</strong>..................................................................................................33<br />
3.5.1. Simultaneous Determination <strong>of</strong> Association Constants and <strong>Protein</strong> Activity.............33<br />
3.5.2. The Gibbs Free Energy <strong>of</strong> Binding................................................................................35<br />
3.5.3. Osmotic Stress and Electrostatic <strong>Interaction</strong>s...............................................................35<br />
3.5.3.1. Davies-Stockes-Robinson Electrostatic Screening Approach .................................35<br />
3.5.3.2. Wyman Linkage <strong>Analysis</strong> .........................................................................................36<br />
3.5.4. Protonation Equilibria ....................................................................................................37<br />
3.5.5. <strong>Thermodynamic</strong>s ............................................................................................................38<br />
3.5.5.1. Coupling Between Binding and Conformational Transition <strong>of</strong> Ligand..................38<br />
3.5.5.2. Enthalpy-Entropy Compensation in Congener Series – General Model 199 .............39<br />
3.6. Isothermal Titration Calorimetry.......................................................................................40<br />
3.6.1. Calorimetric Measurements for m 7 GpppG....................................................................41<br />
3.6.1.1. Modified "Single Injection" Experiment ..................................................................41<br />
3.6.2. Calorimetric Data Treatment .........................................................................................42<br />
3.6.2.1. Buffer Ionization Heats..............................................................................................42<br />
3.7. NMR Spectroscopy................................................................................................................42<br />
3.7.1. NMR Spectra Recording................................................................................................43<br />
3.7.2. NMR Data <strong>Analysis</strong> .......................................................................................................44<br />
3.7.2.1. Concentration Dependence........................................................................................44<br />
3.7.2.2. Temperature Dependence..........................................................................................44<br />
3.8. Dynamic Light Scattering ....................................................................................................45<br />
3.9. Statistical <strong>Analysis</strong>.................................................................................................................46<br />
3.10. Additional Information − Crystallography...................................................................47<br />
4. RESULTS AND DISCUSSION..................................................................................... 48<br />
4.1. Methodological Aspects <strong>of</strong> Studies <strong>of</strong> Non-Enzymatic <strong>Protein</strong>.......................................48<br />
4.1.1. Solution <strong>of</strong> Spectroscopic Difficulties...........................................................................49<br />
4.1.2. Activity <strong>of</strong> Non-Enzymatic <strong>Protein</strong>...............................................................................51<br />
4.1.3. Incorrectness <strong>of</strong> Data Linearization in Case <strong>of</strong> Strong <strong>Interaction</strong>s ............................58<br />
4.2. Molecular Mechanism <strong>of</strong> Recognition <strong>of</strong> <strong>mRNA</strong> 5' Cap Structure by <strong>eIF4E</strong> Cap-<br />
Binding <strong>Protein</strong> ...................................................................................................................................60<br />
4.2.1. Affinity <strong>of</strong> Cap Analogues for <strong>eIF4E</strong>............................................................................60<br />
4.2.1.1. Equilibrium Binding Constants.................................................................................60<br />
4.2.1.2. Binding Affinity vs Inhibitory Properties <strong>of</strong> Cap Analogues ..................................62<br />
4.2.2. Parsing the Free Energy <strong>of</strong> <strong>eIF4E</strong> Binding to <strong>mRNA</strong> 5' Cap.......................................64<br />
4.2.2.1. Energetic Cost <strong>of</strong> 7-Substituent Alteration...............................................................66<br />
4.2.2.2. General Separation <strong>of</strong> the Binding Free Energy.......................................................68<br />
4.2.2.3. Relation <strong>of</strong> Stacking - Hydrogen Bonding Cooperativity to Other Cap-Binding<br />
Molecules ....................................................................................................................................69<br />
4.2.2.4. Energetic Cost <strong>of</strong> Methylation <strong>of</strong> the N(2)-Amino Group.......................................70<br />
VI
4.2.2.5. Energetic Cost <strong>of</strong> the Second Nucleoside Addition and Modification ................... 70<br />
4.2.2.6. Contributions <strong>of</strong> Phosphate Groups and Trapped Water Molecules....................... 72<br />
4.2.3. <strong>Interaction</strong>s in Context <strong>of</strong> Environment........................................................................ 73<br />
4.2.3.1. <strong>Interaction</strong> <strong>of</strong> <strong>eIF4E</strong>-Cap in the Presence <strong>of</strong> Electrolyte ......................................... 74<br />
4.2.3.1.1. Davies-Stockes-Robinson Electrostatic Screening Approach........................... 74<br />
4.2.3.1.1.1. Electrostatic Effects ...................................................................................... 74<br />
4.2.3.1.1.2. Two-Step Mechanism <strong>of</strong> the Cap-<strong>eIF4E</strong> Association ............................. 77<br />
4.2.3.1.1.3. Relation to the "Clamping Cycle" ............................................................ 78<br />
4.2.3.1.1.4. Osmotic Stress............................................................................................... 78<br />
4.2.3.1.2. Wyman Linkage <strong>Analysis</strong> ................................................................................... 79<br />
4.2.3.1.2.1. Electrolyte Effect........................................................................................... 79<br />
4.2.3.1.2.2. Osmotic Stress............................................................................................... 80<br />
4.2.3.1.3. Comparison <strong>of</strong> the Two Approaches .................................................................. 80<br />
4.2.3.1.4. Reasons for KCl-Dependence <strong>of</strong> Internal Rearrangement Rate Constants....... 80<br />
4.2.3.1.5. Discussion <strong>of</strong> Hydration Effects ......................................................................... 81<br />
4.2.3.1.6. Conformational Change <strong>of</strong> <strong>eIF4E</strong> upon Cap Binding........................................ 82<br />
4.2.3.1.6.1. Deaggregation <strong>of</strong> <strong>eIF4E</strong> Induced by Cap Binding ...................................... 82<br />
4.2.3.2. Ionic Equilibria in <strong>eIF4E</strong>-Cap Complex................................................................... 83<br />
4.2.4. Conclusions .................................................................................................................... 85<br />
4.3. Molecular <strong>Interaction</strong>s <strong>with</strong>in Ternary Complexes Involving <strong>eIF4E</strong>, a Cap Analogue,<br />
and an <strong>eIF4E</strong>-Binding Motif from eIF4G or 4E-BP1 <strong>Protein</strong>s.................................................... 86<br />
4.3.1. <strong>eIF4E</strong> Fluorescence Quenching Pattern upon Peptide Binding................................... 87<br />
4.3.2. Cooperativity in Ternary Peptide-<strong>eIF4E</strong>-Cap Complexes ........................................... 90<br />
4.3.2.1. Cooperativity <strong>of</strong> Cap and eIF4G Peptides Binding.................................................. 91<br />
4.3.2.2. Cooperativity <strong>of</strong> Cap and 4E-BP1 Peptides Binding............................................... 93<br />
4.3.3. Regulation <strong>of</strong> 4E-BP1 Activity by Phosphorylation .................................................... 94<br />
4.3.4. Concluding Remarks...................................................................................................... 96<br />
4.4. <strong>Thermodynamic</strong>s <strong>of</strong> <strong>mRNA</strong> 5' Cap Binding by <strong>eIF4E</strong> .................................................... 97<br />
4.4.1. Van't H<strong>of</strong>f <strong>Analysis</strong> <strong>of</strong> Binding <strong>of</strong> <strong>eIF4E</strong> to Cap Analogues....................................... 97<br />
4.4.2. Direct Calorimetric Measurements <strong>of</strong> Binding Enthalpy........................................... 106<br />
4.4.3. Protonation Equilibrium <strong>of</strong> m 7 GpppG ........................................................................ 108<br />
4.4.4. Conformational Equilibrium <strong>of</strong> Cap Analogues......................................................... 110<br />
4.4.5. Could the Positive Values <strong>of</strong> ΔCp° Result from <strong>Protein</strong> Deaggregation ? ................ 113<br />
4.4.6. Enthalpy-Entropy Compensation for Individual Cap Analogues .............................. 113<br />
4.4.7. Biological Implications <strong>of</strong> Non-Linear van't H<strong>of</strong>f <strong>Thermodynamic</strong>s........................ 116<br />
4.4.8. Isothermal Enthalpy-Entropy Compensation in Congener Series ............................. 117<br />
4.4.8.1. Molecular Interpretation <strong>of</strong> Isothermal Enthalpy-Entropy Compensation ........... 119<br />
4.4.9. Inquiries about Origins <strong>of</strong> the Unusual Positive Heat Capacity Change................... 121<br />
4.4.9.1. Changes <strong>of</strong> Solvent Accessible Surfaces Upon Binding ....................................... 122<br />
4.4.9.2. Electrostatic Contributions to Heat Capacity Changes.......................................... 123<br />
4.4.9.3. Tryptophan Stacking <strong>with</strong> Cationic 7-Methylguanosine Moiety .......................... 124<br />
4.4.9.4. Coupling <strong>of</strong> Other Intermolecular Equilibria to <strong>eIF4E</strong> – Cap Binding................. 127<br />
4.4.10. Linear Correlation between ΔCp° and ΔG°................................................................. 128<br />
4.4.11. Discussion <strong>with</strong> Other Authors.................................................................................... 130<br />
4.4.12. Conclusions .................................................................................................................. 131<br />
5. SUMMARY................................................................................................................... 133<br />
6. REFERENCES............................................................................................................. 135<br />
VII
Abbreviations<br />
used throughout the text:<br />
4E-BP 4E binding protein;<br />
A absorbance;<br />
Å angström, 10 -10 m;<br />
bz 7 GTP 7-benzylguanosine 5’-triphosphate;<br />
CPK colours scheme developed by Corey, Pauling and Koltun: carbon - light grey;<br />
oxygen – red; hydrogen – white; nitrogen - light blue; phosphorus –<br />
orange.<br />
cx complex;<br />
DLS Dynamic Light Scattering;<br />
DNA deoxyribonucleic acid;<br />
DTT dithiothreitol;<br />
EDTA disodium ethylenediaminetetraacetate;<br />
eIF eukaryotic initiation factor;<br />
ESI MS Electrospray Ionization Mass Spectrometry;<br />
et 7 GTP 7-ethylguanosine 5’-triphosphate;<br />
F observed fluorescence;<br />
F0<br />
calculated initial fluorescence;<br />
GDP guanosine 5’-diphosphate;<br />
GMP guanosine 5’-monophosphate;<br />
GpppG P 1 -guanosine-5’ P 3 -guanosine-5’ triphosphate;<br />
GTP guanosine 5’-triphosphate;<br />
Hepes N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid];<br />
HPLC High Performance Liquid Chromatography;<br />
I ionic strength;<br />
ITC Isothermal Titration Calorimetry;<br />
k Boltzmann constant, 1.38 ⋅ 10 -23 J⋅K -1 ;<br />
K observed association constant for stacking, determined for individual<br />
protons from NMR signals;<br />
Kas<br />
Kas<br />
observed association constant;<br />
(micro)<br />
Kas<br />
observed microscopic association constant;<br />
pH-ind<br />
calculated, hypothetical association constant for binding <strong>of</strong> two<br />
species in the ionic states optimal for binding;<br />
Kt<br />
thermodynamic association constant;<br />
kDa kilodalton, 1000 atomic mass units;<br />
1K self-stacking/unstacking equlibrium constant;<br />
L ligand;<br />
m2 2,7 GTP N 2 ,7-dimethylguanosine 5’-triphosphate;<br />
m3 2,2,7 GTP N 2 ,N 2 ,7-trimethylguanosine 5’-triphosphate;<br />
m 7 G 7-methylguanosine;<br />
m 7 GDP 7-methylguanosine 5’-diphosphate;<br />
m 7 GMP 7-methylguanosine 5’-monophosphate;<br />
m 7 Gpppm 2’O G P 1 -7-methylguanosine-5’ P 3 -2'-O-methylguanosine-5’ triphosphate;<br />
m 7 GpppA P 1 -7-methylguanosine-5’ P 3 -adenosine-5’ triphosphate;<br />
m 7 GpppC P 1 -7-methylguanosine-5’ P 3 -cytosine-5’ triphosphate;<br />
VIII
m 7 GpppG P 1 -7-methylguanosine-5’ P 3 -guanosine-5’ triphosphate;<br />
m 7 Gpppm 7 G P 1 -7-methylguanosine-5’ P 3 -7-methylguanosine-5’ triphosphate;<br />
m 7 GppppG P 1 -7-methylguanosine-5’ P 3 -guanosine-5’ tetraphosphate;<br />
m 7 Gppppm 7 G P 1 -7-methylguanosine-5’ P 3 -7-methylguanosine-5’ tetraphosphate;<br />
m 7 GTP 7-methylguanosine 5’-triphosphate;<br />
M molar concentration (1 M = 1 mol⋅dm -3 );<br />
MALDI-TOF MS Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass<br />
Spectrometry;<br />
<strong>mRNA</strong> messenger ribonucleic acid;<br />
NMR Nuclear Magnetic Resonance;<br />
NMWL nominal molecular weigh limit;<br />
P probability that distribution <strong>of</strong> fitting residuals is random;<br />
p, PO3 - ; phosphate group;<br />
p-Cl-bz 7 GTP 7-(p-chlorobenzyl)-guanosine 5’-triphosphate;<br />
Pact<br />
active protein;<br />
PDB <strong>Protein</strong> Data Bank (http://rcsb.icm.edu.pl/);<br />
pH -log10 <strong>of</strong> H + activity approximated by concentration;<br />
pHopt<br />
pKa<br />
optimal pH value;<br />
-log10 <strong>of</strong> H + acidic dissociation constant;<br />
pKL<br />
-log10 from the effective acidic dissociation constant <strong>of</strong> a ligand;<br />
pKP<br />
-log10 from the effective acidic dissociation constant <strong>of</strong> a protein<br />
residue;<br />
P(ν1,ν2) probability that improvement <strong>of</strong> the fit <strong>with</strong> the smaller number <strong>of</strong><br />
degrees <strong>of</strong> freedom (ν2) in comparison <strong>with</strong> that <strong>with</strong> the greater<br />
number (ν2) is random;<br />
R universal gas constant, 8.3143 J⋅mol -1 ⋅K -1 ;<br />
r 2<br />
linear correlation coefficient;<br />
R 2<br />
goodness <strong>of</strong> fit;<br />
RNA ribonucleic acid;<br />
SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis;<br />
t temperature in °C;<br />
T absolute temperature in K;<br />
TH<br />
temperature at which ΔH° = 0;<br />
TS<br />
temperature at which ΔS° = 0;<br />
[x] molar concentration <strong>of</strong> x expressed in M (1 M = 1 mol⋅dm -3 );<br />
° denotes standard state; the hypothetical state <strong>of</strong> solute at the standard<br />
concentration (1 mol⋅dm -3 ) and at arbitrarily chosen temperature <strong>of</strong><br />
293 K (20 °C), and exhibiting infinitely dilute solution behaviour; ∗<br />
ε molar extinction coefficient;<br />
δ chemical shift;<br />
ΔCp° standard molar heat capacity change at constant pressure;<br />
ΔCp°sst<br />
contribution to standard molar heat capacity change, resulting from<br />
thermodynamic coupling <strong>with</strong> self-stacking;<br />
Δδ change <strong>of</strong> chemical shift (Δδ = δ(mixture) − δ(free));<br />
ΔG° standard molar Gibbs free energy change (observed);<br />
∗ according to IUPAC Compendium <strong>of</strong> Chemical Terminology, 2 nd Edition (1997)<br />
IX
ΔG°el<br />
contribution <strong>of</strong> electrolyte effect to standard molar Gibbs free energy<br />
<strong>of</strong> complex stabilization;<br />
ΔG° pH-ind<br />
standard molar Gibbs free energy <strong>of</strong> binding <strong>of</strong> two species in the<br />
ionic states that are optimal for binding;<br />
ΔG°L<br />
standard molar Gibbs free energy change <strong>of</strong> partial protonation <strong>of</strong> a<br />
ligand at a given pH;<br />
ΔG°P<br />
standard molar Gibbs free energy change <strong>of</strong> partial deprotonation <strong>of</strong> a<br />
protein residue at a given pH;<br />
ΔH° standard molar enthalpy change;<br />
ΔH°0<br />
intrinsic standard molar enthalpy change;<br />
ΔH°cal<br />
calorimetric standard molar enthalpy change;<br />
ΔH°ion<br />
standard molar enthalpy <strong>of</strong> buffer ionization at given pH;<br />
ΔH°sst<br />
contribution to standard molar enthalpy change, resulting from<br />
thermodynamic coupling <strong>with</strong> self-stacking;<br />
ΔH°vH<br />
standard molar van't H<strong>of</strong>f enthalpy change;<br />
1ΔH° standard molar enthalpy change <strong>of</strong> self-stacking;<br />
ΔN number <strong>of</strong> exchanged water molecules;<br />
ΔS° standard molar entropy change;<br />
ΔS°0<br />
intrinsic standard molar entropy change;<br />
ΔS°el<br />
contribution <strong>of</strong> electrolyte effect to standard molar entropy change;<br />
ΔS°sst<br />
contribution to standard molar entropy change, resulting from<br />
thermodynamic coupling <strong>with</strong> selfsatcking;<br />
1ΔS° standard molar entropy change <strong>of</strong> self-stacking;<br />
λem<br />
emission wavelength;<br />
excitation wavelength.<br />
λex<br />
X
Preface<br />
The thesis concerns the rules <strong>of</strong> intermolecular recognition <strong>of</strong> <strong>mRNA</strong> 5' terminus by<br />
eukaryotic translation initiation <strong>eIF4E</strong> factor and thermodynamic origins <strong>of</strong> the complex<br />
stability. Molecular spectroscopy and isothermal calorimetry were used to address the<br />
mechanistic bases <strong>of</strong> the process <strong>of</strong> association. Conclusions resulting from the<br />
experimental data were discussed in context <strong>of</strong> biological processes in the living cell and <strong>of</strong><br />
general thermodynamic features <strong>of</strong> protein-ligand interactions.<br />
The work is composed <strong>of</strong> the following chapters:<br />
1. INTRODUCTION which contains a concise and far from exhaustive survey <strong>of</strong> the<br />
present knowledge about significance <strong>of</strong> the studied objects in biology and medicine,<br />
and points to some selected thermodynamic aspects <strong>of</strong> macromolecular interactions,<br />
that are fundamental but not always explicitly accented in literature;<br />
2. AIM OF THE WORK defines the context and the particular goals <strong>of</strong> the thesis;<br />
3. MATERIALS AND METHODS – provide necessary information about chemicals,<br />
syntheses, applied equipment, experimental protocols, and procedures <strong>of</strong> data<br />
analysing;<br />
4. RESULT AND DISCUSSION are merged so that the results can be immediately<br />
interpreted in light <strong>of</strong> other phenomena or literature data, <strong>with</strong>out perpetual referring to<br />
different chapters. The chapter addresses four main problems:<br />
4.1. Methodological aspects <strong>of</strong> studies <strong>of</strong> non-enzymatic proteins;<br />
4.2. Molecular mechanism <strong>of</strong> recognition <strong>of</strong> <strong>mRNA</strong> 5' cap structure by <strong>eIF4E</strong>;<br />
4.3. Molecular interactions <strong>with</strong>in ternary complexes that consist on <strong>eIF4E</strong>, a cap<br />
analogue, and additionally involve an <strong>eIF4E</strong>-binding motif from either the<br />
initiation factor 4G (eIF4G) or the translation inhibitor (4E-BP1). The former is<br />
represented by two is<strong>of</strong>orms (eIF4GI and eIF4GII), and the latter is studied as the<br />
unphosphorylated, monophosphorylated, and diphosphorylated species.<br />
4.4. <strong>Thermodynamic</strong>s <strong>of</strong> <strong>mRNA</strong> 5' cap binding to <strong>eIF4E</strong> and its possible consequences<br />
for biological activity.<br />
5. SUMMARY recollects the main conclusions drawn from the studies.<br />
The results presented in this work were partially published, 1-7 some <strong>of</strong> them are in<br />
press (Chapter 4.4.9.3.) or in preparation for submission (data from 4.1. and 4.4.).<br />
XI
1. Introduction<br />
1.1. Brief Description <strong>of</strong> Biological Aspects<br />
Life <strong>of</strong> organisms depends on delicate balance <strong>of</strong> many biochemical processes. Such<br />
balance needs sensitive and efficient regulatory mechanisms to coordinate growth,<br />
development and reproduction. The most fundamental mechanisms are related to gene<br />
expression.<br />
1.1.1. Central Dogma <strong>of</strong> Molecular Biology<br />
The Central Dogma was first proposed by F. Crick in 1958, and states that genetic<br />
information flow is unidirectional, from DNA to protein via an RNA intermediate. 8 In<br />
order to be expressed, genetic information encoded in DNA has first to be transcribed into<br />
a complementary copy <strong>of</strong> RNA. There are some exceptions from this rule, i. e. reverse<br />
transcriptases <strong>of</strong> retroviruses synthesize DNA from an RNA template, and RNA viruses<br />
such as poliovirus or influenza virus apply RNA replication. 9<br />
DNA replication<br />
DNA<br />
Reverse transcription<br />
Transcription<br />
(RNA synthesis)<br />
Scheme 1-1. Central Dogma <strong>of</strong> Molecular Biology.<br />
RNA transcripts are either used directly as transfer RNAs (tRNAs), ribosomal RNAs<br />
(rRNAs), and small nuclear RNAs (snRNAs) or, as messenger RNAs (<strong>mRNA</strong>s), are<br />
translated into proteins. The <strong>mRNA</strong> transcripts are cotranscriptionally processed by<br />
capping (linkage <strong>of</strong> 7-methylguanosine by a 5' - 5' triphosphate bridge to the first<br />
transcribed nucleoside), splicing (intron excision), and polyadenylation <strong>of</strong> the 3'<br />
terminus. 10 The mature <strong>mRNA</strong> is transported into the cytoplasm (except for mitochondrial<br />
or chloroplast protein <strong>mRNA</strong>s). In eukaryotes, transcripts contain one sequence encoding a<br />
1<br />
<strong>mRNA</strong> PROTEIN<br />
RNA<br />
replication<br />
Translation initiation<br />
Translation<br />
(protein synthesis)
polypeptide, but some sequences can yield more than one polypeptide as a consequence <strong>of</strong><br />
alternative transcriptional starting sites and/or alternative splicing. Translation occurs on<br />
ribosomes. The ribosome is made up <strong>of</strong> two nonidentical subunits, the large (60S) and<br />
small one (40S). Each <strong>of</strong> them contains one or more rRNA molecules and 20-50 different<br />
ribosomal proteins. Several ribosomes may simultaneously translate the same <strong>mRNA</strong><br />
molecule. <strong>Protein</strong>s are synthesized starting <strong>with</strong> their N termini, corresponding to<br />
translation <strong>of</strong> the <strong>mRNA</strong> in the 5' to 3' direction. Translation may be divided into three<br />
stages: initiation, elongation, and termination. Some proteins can fold to their native<br />
conformation during elongation <strong>of</strong> the peptide chain, i. e. before the synthesis is<br />
completed, or it is only after that, <strong>of</strong>ten <strong>with</strong> help <strong>of</strong> other proteins, molecular chaperones.<br />
<strong>Protein</strong>s can undergo many post-translational covalent modifications, e. g. proteolysis,<br />
protein splicing, phosphorylation, and disulphide bond formation, yielding finally the<br />
biologically active form. 11<br />
1.1.2. <strong>Protein</strong> Functions<br />
<strong>Protein</strong>s participate in almost every biological process. Exemplary functions, which<br />
proteins are responsible for, are as follows: structure building, metabolism, transport<br />
throughout body, storage <strong>of</strong> ions, membrane transport, muscle contraction, osmotic<br />
regulation, immunological defence, cell recognition, generation and transduction <strong>of</strong><br />
nervous signals, and control functions. 9 Control factors are even by orders <strong>of</strong> magnitude<br />
less abundant than structural proteins or enzymes, but they perform superior functions in<br />
the living cell, regulating abundancies and activities <strong>of</strong> the remaining proteins. <strong>Protein</strong><br />
control factors act as hormones and fulfil control on gene expression, cell growth,<br />
proliferation, and differentiation.<br />
One <strong>of</strong> the eukaryotic regulatory proteins is <strong>eIF4E</strong>, initiation translation factor 4E,<br />
the key protein in translational control <strong>of</strong> biosynthesis. 12<br />
1.1.3. Translation Initiation<br />
Translation initiation is a complex (Scheme 1-2), rate-limiting step <strong>of</strong> gene<br />
expression. 13 During initiation, the correct starting site on <strong>mRNA</strong> is identified and binding<br />
<strong>of</strong> the ribosome occurs. All <strong>mRNA</strong>s and snRNAs that are transcribed in the nucleus<br />
possess a 5'-terminal "cap", m 7 GpppN, in which 7-methylguanosine is linked by a 5’-5’-<br />
triphosphate bridge to the first nucleoside 14 (Scheme 1-3). The cap structure participates in<br />
2
Scheme 1-2. Eukaryotic translation initiation pathway. Initiation factors (eIF) are shown as colourcoded<br />
labelled circles, and appear as rectangulars when first implicated in the pathway. 60S, 40S -<br />
large and small ribosomal subunits, respectively; Met – initiator methionyl-tRNA; m7G AUG -<br />
<strong>mRNA</strong> <strong>with</strong> the 7-methylguanosine cap and the starting codon (AUG). <strong>eIF4E</strong> is responsible for<br />
recognition <strong>of</strong> the <strong>mRNA</strong> cap and mediates joining <strong>of</strong> the remaining eIFs, which finally leads to<br />
ribosome recruitment and positioning. Scheme adopted from Hershey and Merrick. 15<br />
3
the splicing <strong>of</strong> <strong>mRNA</strong> precursors, 16,17 is necessary for RNA nuclear export 18-20 and for<br />
optimal <strong>mRNA</strong> translation, 14,21 and affects <strong>mRNA</strong> stability. 19 After nuclear export to the<br />
cytosol, the cap <strong>of</strong> snRNAs is further methylated at the amino group <strong>of</strong> the guanosine<br />
moiety, forming m GpppN<br />
7 , 2 , 2<br />
22<br />
This trimethylated structure is responsible for import <strong>of</strong><br />
3<br />
the snRN-protein spliceosomal complexes (snRNPs) back into the nucleus, 23,24 where<br />
snRNPs take part in pre-<strong>mRNA</strong> splicing. 25 A variety <strong>of</strong> <strong>mRNA</strong> cap structures can be<br />
present in the cytoplasm, and most <strong>of</strong> them are additionally methylated at 2'O <strong>of</strong> the second<br />
ribose moiety: m 7 Gpppm2 6,2'O A, m 7 Gpppm 2'O G, m 7 Gpppm 2'O A, m 7 Gpppm 2'O C, m 7 GpppA,<br />
and m 7 GpppG. 26 The original <strong>mRNA</strong> cap can be replaced by the trimethylguanosine cap<br />
structure as a result <strong>of</strong> trans-splicing which occurs in primitive animals, 27,28 e. g. in the C.<br />
elegans nematode about 70% <strong>of</strong> the <strong>mRNA</strong> population contains GpppN<br />
4<br />
m 7 , 2 , 2<br />
3<br />
Regular translation initiation is cap-dependent, though there are also two alternative<br />
ways to recruit the 40S ribosome subunit to the <strong>mRNA</strong>, mediated by an internal ribosome<br />
entry site (IRES) or by the poly(A) tail. 30 The cap function in translation initiation is<br />
mediated by the eukaryotic initiation factor 4E (<strong>eIF4E</strong>), 12 which is the smallest subunit <strong>of</strong><br />
the heterotrimeric eIF4F initiation complex. 31 eIF-4F appears to be also required for the<br />
cap-independent translation <strong>of</strong> naturally uncapped <strong>mRNA</strong>s. 32 In higher eukaryotes eIF4F<br />
consists <strong>of</strong> <strong>eIF4E</strong>, eIF4A, an RNA-dependent ATPase and RNA helicase, and eIF4G, a<br />
multifunctional, adaptor protein. 30,31,33 Two is<strong>of</strong>orms <strong>of</strong> eIF4G, namely eIF4GI (171<br />
kDa) 34 and eIF4GII (176 kDa) 35 are present in mammals. These scaffolding molecules act<br />
as a molecular bridge between <strong>eIF4E</strong> and eIF4A. 33,35 The eIF4Gs interact <strong>with</strong> the<br />
ribosome-bound multiprotein factor eIF3, to recruit the 40S ribosomal subunit. Additional<br />
interaction <strong>of</strong> the eIF4Gs <strong>with</strong> the poly(A)-binding protein (PABP) allows <strong>mRNA</strong><br />
circularization 30,31 and ribosome recycling. 36<br />
* H<br />
N<br />
H 2<br />
O<br />
N 6<br />
1<br />
2<br />
3<br />
N<br />
CH 3<br />
N<br />
7<br />
+<br />
9<br />
N<br />
8<br />
H<br />
H<br />
H<br />
H<br />
OH<br />
HO<br />
O<br />
CH 2<br />
H<br />
O O O<br />
O P O P O P O<br />
O - O - O -<br />
Scheme 1-3. Chemical structure <strong>of</strong> eukaryotic <strong>mRNA</strong> 5' terminus called "cap". Proton that partially<br />
dissociates at physiological pH is marked <strong>with</strong> asterisk.<br />
CH 2<br />
H<br />
H<br />
O<br />
O<br />
H<br />
Base<br />
H<br />
. 29<br />
OH O-H or -CH3 <strong>mRNA</strong> chain
1.1.4. Eukaryotic Initiation Factor 4E<br />
The eukaryotic initiation factor 4E (<strong>eIF4E</strong>) is a 25 kDa protein (217 amino acids)<br />
which plays a pivotal role in translational control. <strong>eIF4E</strong> is a cytoplasmic protein, and acts<br />
as a subunit <strong>of</strong> the eIF4F initiation complex. However, a fraction <strong>of</strong> <strong>eIF4E</strong> localizes also in<br />
the nucleus 37 and participates in nucleocytoplasmic transport <strong>of</strong> specific, growth regulatory<br />
<strong>mRNA</strong>. 38 Since <strong>eIF4E</strong> colocalizes in nuclear speckles <strong>with</strong> splicing factors, it is also<br />
supposed to be involved in splicing and 3' <strong>mRNA</strong> processing. 39 All these additional<br />
functions <strong>of</strong> <strong>eIF4E</strong> appear to be dependent on its intrinsic ability to recognize the <strong>mRNA</strong> 5'<br />
cap. Thus, this single biochemical activity <strong>of</strong> <strong>eIF4E</strong> is versatile enough to play roles in<br />
divergent processes in different cellular compartments. The shuttling protein, <strong>eIF4E</strong>-<br />
transporter (4E-T), that mediates the nuclear import <strong>of</strong> <strong>eIF4E</strong> via the α/β importin pathway<br />
by a piggy-back mechanism was discovered recently. 40<br />
<strong>eIF4E</strong> is the least abundant protein initiation factor in the cell (0.8 ⋅ 10 6 molecules<br />
per 1 HeLa cell, i. e. only 0.021 % <strong>of</strong> cell protein), 41 and its accessibility for formation <strong>of</strong><br />
the initiation eIF4F heterocomplex is supposed to regulate the overall efficiency <strong>of</strong><br />
ribosome recruitment. 31,42 Expression <strong>of</strong> <strong>eIF4E</strong> is regulated at the transcription level. 43 On<br />
the other hand, <strong>eIF4E</strong> is a very instable protein; its instability index calculated on the basis<br />
<strong>of</strong> the sequence 44 is 52.17 for human full length <strong>eIF4E</strong> and 47.41 for murine protein<br />
(residues 28-217) (Table 1-1). This biochemical property may account for the low<br />
abundance <strong>of</strong> <strong>eIF4E</strong> in the cell. <strong>Interaction</strong> between <strong>eIF4E</strong> and eIF4G upon eIF4F complex<br />
formation is inhibited by 4E-binding proteins (mammalian 4E-BP1, 4E-BP2, 4E-BP3, 31<br />
and yeast p20 45 ).<br />
Table 1-1. Comparison <strong>of</strong> protein stabilities resulting from frequency <strong>of</strong> occurrence <strong>of</strong> particular<br />
dipeptides in the amino acid sequences. 44<br />
<strong>Protein</strong> (Organism) Instability Index<br />
<strong>eIF4E</strong> (residues 1-217; human) 52.17<br />
<strong>eIF4E</strong> (residues 28-217; mouse) 47.41<br />
stability limit 40<br />
BPTI (bovine) 32.60<br />
S100 (human) 25.22<br />
PNP (E. coli) 23.13<br />
lysozyme (mouse) 17.76<br />
5
Figure 1-1. Sequence alignments <strong>of</strong> <strong>eIF4E</strong> from mammalia. The relative accessibility <strong>of</strong> each<br />
protein residue was extracted from DSSP 46 and PHD 47 files and shown as colour boxes: white –<br />
buried; cyan – intermediate accessible; blue – accessible; blue <strong>with</strong> red borders – exposed into<br />
solvent; red – unstructured lysine side chains; gap – unstructured main chain. The secondary<br />
structural elements were assigned from the X-ray structures <strong>of</strong> human <strong>eIF4E</strong> in complex <strong>with</strong><br />
m 7 GpppA (1IPB.pdb 48 , top) and murine <strong>eIF4E</strong> <strong>with</strong> m 7 GpppG (1L8B 1 , bottom); α - alpha helices;<br />
η - 310 helices; β - beta strands; TT - strict beta turns.<br />
Table 1-2. Characteristics <strong>of</strong> <strong>eIF4E</strong> sequence conservation among organisms<br />
Group <strong>of</strong> Organisms (<strong>Protein</strong> Is<strong>of</strong>orm) Identity (%) Similarity (%)<br />
Mammalia 98 99<br />
Vertebrates 88 95<br />
Animals a<br />
19 49<br />
Plants (<strong>eIF4E</strong>-1) 55 69<br />
Plants (<strong>eIF4E</strong>-2) 52 69<br />
Plants b<br />
26 53<br />
Fungi c<br />
28 50<br />
All eukaryotes (<strong>eIF4E</strong>-1) 10 35<br />
All eukaryotes d<br />
5 23<br />
Human vs Yeast 29 44<br />
a including only <strong>eIF4E</strong>-1 from Caenorhabditis elegans<br />
b including both <strong>eIF4E</strong>-1 and <strong>eIF4E</strong>-2 is<strong>of</strong>orms<br />
b including only <strong>eIF4E</strong>-1 from Schizosaccharomyces pombe<br />
c including all is<strong>of</strong>orms <strong>of</strong> <strong>eIF4E</strong> (33 sequences)<br />
6
Figure 1-2 (in the previous page). Sequence alignments <strong>of</strong> <strong>eIF4E</strong> (or <strong>eIF4E</strong>-1 if more is<strong>of</strong>orms<br />
exist) from various organisms: human 49 , mouse 50 , rabbit 51 , Xenopus laevis 52 , Aplysia californica 53 ,<br />
Drosophila melanogaster 54 , Caenorhabditis elegans 55 , maize 56 , rice 57 , wheat 58 , Arabidopsis<br />
thaliana 59 , Candida glabrata 60 , Saccharomyces cerevisiae 61 , Schizosaccharomyces pombe 62 . The<br />
sequences are separated in groups corresponding to the animals (1), plants (2) and fungi kingdoms<br />
(3). The alignments were done by the ClustalW program 63 and are colour coded according to the<br />
following rules: red box, white character - strict identity; red character - similarity in a group (><br />
70% <strong>of</strong> residues are similar according to physico-chemical properties); blue frame - similarity<br />
across groups; orange box - differences between conserved groups (> 50% <strong>of</strong> residues are<br />
conserved <strong>with</strong>in a group but not conserved from one group to the other). Risler matrix was used<br />
for similarity and difference score calculations 64 . A consensus line indicates as follows: uppercase<br />
– identity; lowercase - consensus level > 0.5; ! is I or V; $ is L or M; % is F or Y; # is N, D, Q or E.<br />
<strong>eIF4E</strong> activity is also regulated by phosphorylation at Ser209 in response to<br />
treatment <strong>of</strong> cells <strong>with</strong> growth factors, hormones, and mitogens. 65-67 Phosphorylation<br />
seems to positively correlate <strong>with</strong> protein synthesis, cell growth and proliferation, 66,68,69 but<br />
the explanation how it could happen at the molecular level remains unclear. 70-74<br />
<strong>eIF4E</strong> is phylogenetically highly conserved (Table 1-2, Fig. 1-1, 1-2) and unusually<br />
rich in tryptophans. Each <strong>of</strong> its eight tryptophans is absolutely conserved among all known<br />
sequences, from fungi to human. Three-dimensional structures <strong>of</strong> murine <strong>eIF4E</strong> bound to<br />
7-methylGDP 75 and 7-methylGpppG, 1 and recently also <strong>of</strong> human <strong>eIF4E</strong> in complex <strong>with</strong><br />
7-methylGTP and 7-methylGpppA 48 were solved by X-ray crystallography. The structure<br />
<strong>of</strong> their yeast homologue bound to 7-methylGDP was established by solution NMR<br />
spectroscopy. 76 Each protein consists <strong>of</strong> one α/β domain, and is shaped like a cupped hand,<br />
<strong>with</strong> the cap analogue located in a narrow cap-binding slot on the concave surface <strong>of</strong> the<br />
protein (Fig. 1-3). Recognition <strong>of</strong> the 7-methylguanine moiety by the human and murine<br />
proteins is mediated by base sandwich-stacking between Trp56 and Trp102, formation <strong>of</strong><br />
Watson-Crick-like hydrogen bonds <strong>with</strong> a side chain carboxylate <strong>of</strong> a conserved Glu103<br />
and a backbone NH <strong>of</strong> Trp102, and a van der Waals contact <strong>of</strong> the N(7)-methyl group <strong>with</strong><br />
Trp166. The phosphate chain forms salt bridges and direct or water-mediated hydrogen<br />
bonds <strong>with</strong> the NH <strong>of</strong> Trp102 and Trp166 indole rings, and side chains <strong>of</strong> Arg112, Lys162,<br />
Arg157, and Lys 206. As regards the second nucleoside, adenosine is stabilized by a direct<br />
hydrogen bond between N 6 -amino group and the backbone carbonyl <strong>of</strong> Thr205. The<br />
second guanosine in invisible in the crystal structure since it is not capable <strong>of</strong> forming this<br />
hydrogen bond. At the same time, the protein loop containing Thr205 is disordered.<br />
8
Figure 1-3. Global fold <strong>of</strong> <strong>eIF4E</strong> bound to a fragment <strong>of</strong> cap, 7-methylguanosine 5’-triphosphate<br />
(shown as sticks in CPK colours). 1 Secondary structure elements are colour coded: yellow - β<br />
strands, magenta - α helices, white - loops and turns. Amino acid belonging to the cap-binding site<br />
are shown as balls and sticks: blue – Lys206, Arg112, Lys162, Arg157 (from the left); purple –<br />
absolutely conserved tryptophans 102, 166, 56 (from the left), red – glutamic acid 103.<br />
1.1.5. Translational Control<br />
To the middle nineties <strong>of</strong> the XX-th century transcription was thought to be the main<br />
subject to a multitude <strong>of</strong> controls. 9 However, the cell applies also a lot <strong>of</strong> regulatory<br />
mechanism at the level <strong>of</strong> translation. Translational control provides the cell <strong>with</strong> benefits<br />
more important than the energetic cost paid for the reaching that step <strong>of</strong> gene expression. 77<br />
The most conspicuous advantage <strong>of</strong> translational control is immediacy. Direct and rapid<br />
intervention gives the cell no time to adjust coupled biochemical reactions and the protein<br />
synthesis is immediately stopped. On the other hand, most translational controls consist in<br />
reversible modifications <strong>of</strong> translation factors, e. g. phosphorylation. The reversibility is<br />
fast and economical in energetic terms, especially for energy-deprived cells. The changes<br />
in transcription rates are considerably greater in magnitude than the changes in translation<br />
9
ates. Thus, while transcription is responsible for the coarse control, translation provides a<br />
means for fine regulation. Moreover, it enables a spatial control <strong>of</strong> protein synthesis, which<br />
plays a key role in effecting synaptic plasticity in the hippocampus. 78 The local protein<br />
synthesis at a stimulated synapse leads to the synapse-specific growth. This, in turn, is<br />
necessary to long-term potentiation that underlies processes <strong>of</strong> learning and memory<br />
encoding. 79,80 Translation gives a possibility to control <strong>of</strong> expression <strong>of</strong> a single gene or <strong>of</strong><br />
whole classes <strong>of</strong> <strong>mRNA</strong>s. For instance, host-cell shut<strong>of</strong>f induced by heat shock 81 or by<br />
picornaviruses, 82 as well as retroviral gene expression (such as human immunodeficiency<br />
virus type 1, HIV-1 83 ) are controlled at the translational level. Translational controls are<br />
common for regulation <strong>of</strong> gene expression during development (e. g. in oocytes), and in<br />
other systems lacking transcriptional control. Finally, translational control has proven to be<br />
an important factor in human cancer. 84-87<br />
1.1.5.1. Regulation <strong>of</strong> eIF4F Complex Formation<br />
The accessibility <strong>of</strong> <strong>eIF4E</strong> in mammals is regulated by interactions <strong>with</strong> small<br />
translational repressors, <strong>eIF4E</strong>-binding proteins, 4E-BP1, 4E-BP2 and 4E-BP3, which<br />
prevent productive interactions between <strong>eIF4E</strong> and eIF4G. 88,89 They also inhibit<br />
phosphorylation <strong>of</strong> <strong>eIF4E</strong> at Ser 209. 68<br />
Sequence analysis <strong>of</strong> the 4E-BPs and eIF4Gs suggests that these otherwise unrelated<br />
protein families have converged on the same <strong>eIF4E</strong> binding strategy. 45,89 The <strong>eIF4E</strong><br />
binding site shared between the 4E-BPs and the eIF4Gs is BXXYDRXFLΦ, where B is a<br />
conserved basic residue, X is variable, Φ is a conserved hydrophobic residue, and invariant<br />
residues are shown in boldface. 34,35,67,90 Crystal structures <strong>of</strong> two ternary complexes <strong>of</strong><br />
<strong>eIF4E</strong> <strong>with</strong> 7-methyl-GDP and peptides encompassing the <strong>eIF4E</strong> binding sites, derived<br />
from eIF4GII or 4E-BP1, revealed that both peptides recognize a phylogenetically<br />
invariant, partially hydrophobic, partially acidic surface <strong>of</strong> the convex dorsum <strong>of</strong> <strong>eIF4E</strong> in<br />
the vicinity <strong>of</strong> Trp73, and share a common mode <strong>of</strong> interaction. 91<br />
Some <strong>of</strong> the hypophosphorylated forms <strong>of</strong> 4E-BPs bind to <strong>eIF4E</strong> and these<br />
interactions are highly regulated in cells. 82 Hyperphosphorylation abrogates the interaction<br />
<strong>with</strong> <strong>eIF4E</strong>, which enables recruitment <strong>of</strong> eIF4G, allowing eIF4F complex formation and<br />
translation initiation to proceed. 67,92 The phosphorylation mechanism has been extensively<br />
investigated for 4E-BP1. The protein possesses at least six phosphorylation sites: Thr37,<br />
Thr46, Ser65, Thr70, Ser83 and Ser112 (numbering for the human protein). The ordered,<br />
10
hierarchical model <strong>of</strong> phosphate addition to endogenous 4E-BP1, in which phosphorylation<br />
<strong>of</strong> Thr37 and Thr46 is followed by phosphorylation <strong>of</strong> Thr70 and finally <strong>of</strong> Ser65, 3,92 is<br />
generally accepted, although some points remain controversial. 93-95 At issue is the<br />
influence <strong>of</strong> the single phosphorylation <strong>of</strong> Ser65 or combined Ser65/Thr70<br />
phosphorylation on the 4E-BP1 binding to <strong>eIF4E</strong>. 96<br />
1.1.6. Clinical Aspects <strong>of</strong> the Cellular <strong>eIF4E</strong> Activity Control<br />
1.1.6.1. Relation <strong>of</strong> <strong>eIF4E</strong> Activity to Immunology and Malignancy<br />
Since initiation is a rate-limiting step for translation, it is not surprising that <strong>eIF4E</strong><br />
activity is frequently related to disorder <strong>of</strong> cellular proliferation, growth and<br />
differentiation. 87 The <strong>eIF4E</strong> gene is a proto-oncogene. Overexpression <strong>of</strong> <strong>eIF4E</strong> is caused<br />
by gene amplification. 85 When present in excess <strong>of</strong> the normal physiological amount,<br />
<strong>eIF4E</strong> can stimulate global protein synthesis. 97 Elevated levels <strong>of</strong> <strong>eIF4E</strong> were found in<br />
many cancers, and were shown to stimulate division and cause malignant transformation <strong>of</strong><br />
both normal and immortal cells. 87 Immunological detection <strong>of</strong> <strong>eIF4E</strong> was even reported as<br />
a useful diagnostic tool to identify malignant cells for removal during surgery <strong>of</strong> head and<br />
neck cancer. 98 Affinity <strong>of</strong> <strong>eIF4E</strong> for <strong>mRNA</strong> 5' cap is strongly reduced by the nuclear<br />
promyelocytic leukaemia protein (PML) which mediates suppression <strong>of</strong> oncogenic<br />
transformation and <strong>of</strong> growth. 99<br />
On the other hand, enhanced <strong>eIF4E</strong> activity and its phosphorylation at Ser209 are<br />
engaged in signalling pathways necessary for human peripheral blood T lymphocytes to<br />
progress from the resting state to proliferation and to attain immune competency. 66<br />
1.1.6.2. Relation <strong>of</strong> 4E-BP1 and eIF4G Activities to Human Health and Disease<br />
1.1.6.2.1. Phosphorylation <strong>of</strong> 4E-BP1<br />
4E-BP1 is also known as PHAS-I, phosphorylated heat- and acid-stable protein<br />
regulated by insulin, since insulin causes 4E-BP1 to become hyperphosphorylated and<br />
dissociate from eIF-4E, thus stimulating the overall rate <strong>of</strong> translation and promoting cell<br />
growth. 67<br />
Activation <strong>of</strong> human blood T lymphocytes needs the release <strong>of</strong> <strong>eIF4E</strong> by<br />
hyperphosphorylation <strong>of</strong> 4E-BP1 to enable synthesis <strong>of</strong> proteins that are important for<br />
proliferation. 66<br />
Dissociation <strong>of</strong> 4E-BP1 from <strong>eIF4E</strong> is involved in tumour cell survival. In particular,<br />
11
the α6β4 integrin stimulates the phosphorylation and inactivation <strong>of</strong> 4E-BP1, thus<br />
preventing inhibition <strong>of</strong> <strong>eIF4E</strong>, and finally enhancing translation <strong>of</strong> vascular endothelial<br />
growth factor (VEGF) in human breast carcinoma cells. 100<br />
1.1.6.2.2. Dephosphorylation <strong>of</strong> 4E-BP1 and Cleavage <strong>of</strong> eIF4G<br />
In contrast, heat shock 81 and infection <strong>of</strong> cells <strong>with</strong> picornaviruses, such as poliovirus<br />
and encephalomyocarditis virus, 82 render 4E-BP1 dephosphorylated. Picornaviruses use a<br />
cap-independent mechanism for the translation <strong>of</strong> their RNA, and specific inhibition <strong>of</strong><br />
cap-dependent translation by activation <strong>of</strong> 4E-BP1 is thought to be the major cause <strong>of</strong> a<br />
shut<strong>of</strong>f <strong>of</strong> host protein synthesis in encephalomyocarditis virus-infected cells. Poliovirus<br />
acts doubly, and its second strategy involves also translational control at the initiation<br />
level, since the infection results in the cleavage <strong>of</strong> the eIF4GII protein. 101 The eIF4GII<br />
protein is also a target <strong>of</strong> the rhino and foot-and-mouth-disease viral proteases, 102 while the<br />
HIV-1 viral protease cleaves the eIF4GI protein. 83<br />
Rapid cleavage <strong>of</strong> eIF4GI by caspase-3 occurs also during induced apoptosis. 103,104<br />
1.1.6.3. Anticancer Gene Therapy and Pharmacotherapy<br />
4E-BP1 belongs to a class <strong>of</strong> multiphosphorylated proteins that are dephosphorylated<br />
rapidly after rapamycin treatment. 105 Rapamycin represents a novel family <strong>of</strong> agents <strong>of</strong><br />
antibiotic, antifungal, and, which is the most important, immunosuppressory and<br />
antitumour activity. 106 Rapamycin acts successfully as a cytostatic or induces apoptosis in<br />
malignant cells through inhibition <strong>of</strong> a function <strong>of</strong> mTOR (mammalian target <strong>of</strong><br />
rapamycin) which is a huge, highly conserved protein (~280 kDa). mTOR is a central<br />
controller <strong>of</strong> cell growth and proliferation, influencing an unusually abundant and diverse<br />
set <strong>of</strong> reactions. 107 It acts both as a protein-kinase and protein-phosphatase regulator, and<br />
among others it modulates 4E-BP1 phosphorylation. The mTOR − 4E-BP1 signalling<br />
pathway is important not only for malignancy but also for proliferation in T lymphocytes. 66<br />
The Food and Drug Administration has approved rapamycin in 1999, and the European<br />
Commission in 2000. However, recent data indicate that genetic mutations or<br />
compensatory changes in tumour cells render resistance to rapamycin. 108,109<br />
The important role for translation initiation in the regulation <strong>of</strong> the cell cycle makes<br />
that other components <strong>of</strong> the initiation machinery already begin to be considered attractive<br />
targets for therapeutic intervention. Attention is focused at the activity <strong>of</strong> 4E-BP1, as a<br />
12
crucial mTOR-controlled downstream effector molecule, and at <strong>eIF4E</strong>. The most recent<br />
publications (2002) report that cellular activity <strong>of</strong> <strong>eIF4E</strong> is already now exploited to<br />
selectively kill breast cancer cells during clinical gene therapy. 110 However, there are still<br />
no chemotherapeutic inhibitors <strong>of</strong> the <strong>eIF4E</strong> activity. Thorough biophysical studies <strong>of</strong><br />
functional complexes involving <strong>eIF4E</strong> can make up for this delay and provide a specific<br />
inhibitory agent based on the rationally modified cap structure.<br />
1.2. Some Remarks about <strong>Thermodynamic</strong> Approach to<br />
Molecular <strong>Interaction</strong>s in Solution<br />
Mechanistic insights into molecular bases <strong>of</strong> biological activity do not flow<br />
naturally from the description <strong>of</strong> the crystal structure. The stationary structure does not<br />
reveal all exchange processes that need completing so that the final state, detectable by<br />
crystallography, can come into being. In general, proteins and ligands structures are not<br />
rigid. In particular, non-enzymatic regulatory protein factors are <strong>of</strong>ten very flexible and<br />
experience large and even global conformational changes upon binding to their targets, in<br />
contrast to most <strong>of</strong> enzymes. Such features are characteristic for e. g. transcription factors<br />
that interact <strong>with</strong> DNA. 111 In addition to conformational transitions, formation <strong>of</strong> the<br />
functional macromolecular complexes is accompanied by changes <strong>of</strong> interactions <strong>with</strong> the<br />
environment, first <strong>of</strong> all <strong>with</strong>in the first-layer solvation shell. Hence, the static structural<br />
view needs to be supplemented by thermodynamic description in order to render the<br />
biological functioning on the molecular level comprehensible.<br />
Binding studies <strong>of</strong> specific complexes involving proteins, nucleic acids and small<br />
ligands have proven to be useful to investigate the stabilization energy and the influence <strong>of</strong><br />
solvent on the complex formation, e. g. electrolytic effect, 112-116 water exchange between<br />
the macromolecules and the bulk solvent, 117-121 and linked protonation equilibria. 122-124<br />
1.2.1. <strong>Thermodynamic</strong> and Apparent Association Constant<br />
A thermodynamic equilibrium constant (Kt) which is a function <strong>of</strong> temperature (T)<br />
and pressure (p) only, is expressed in terms <strong>of</strong> equilibrium molar activities (ai): 125<br />
∏ α<br />
νi<br />
t = a i<br />
i=<br />
1<br />
K , Eq. 1-1<br />
where νi are stoichiometric coefficients <strong>of</strong> the reaction and summation ranges by all<br />
species taking part in the reaction. A logarithmic form <strong>of</strong> this relationship:<br />
13
log( K<br />
t<br />
∑ i<br />
i 1<br />
α<br />
=<br />
) = ν ⋅ log( a )<br />
Eq. 1-2<br />
i<br />
makes it possible to analyse easily the dependence <strong>of</strong> Kt on the presence <strong>of</strong> individual<br />
participants <strong>of</strong> the reaction: macromolecules, cations, anions, protons, and water.<br />
An experimentally observed equilibrium association constant (Kas) is defined for<br />
the process converting chosen reactants (e. g. protein (P) and ligand (L)) to products (e. g.<br />
complex (cx)) at a specified set <strong>of</strong> solution conditions, i. e. when activities <strong>of</strong> remaining<br />
reactants (e. g. ions, protons, water molecules) are fixed, e. g.: 118<br />
K<br />
as<br />
[ cx]<br />
= Eq. 1-3<br />
[ P]<br />
⋅[<br />
L]<br />
0<br />
0<br />
This apparent constant is formulated in terms <strong>of</strong> concentrations <strong>of</strong> only a protein, a ligand<br />
and their complex, instead <strong>of</strong> thermodynamic activities <strong>of</strong> all interacting species that could<br />
putatively participate in the reaction. Kas contains involved, hidden information <strong>of</strong> both the<br />
true thermodynamic association constant (Kt) and the changes <strong>of</strong> the activity coefficients<br />
as a function <strong>of</strong> the solution conditions. Therefore, the observed equilibrium binding<br />
constant depends on the environmental variables, i.e. pH, ionic strength and osmolality <strong>of</strong><br />
the solution. 118,119,126 Hence, Kas is a relative quantity. To probe the molecular and<br />
thermodynamic origins <strong>of</strong> stability and specificity <strong>of</strong> <strong>eIF4E</strong> interactions <strong>with</strong> cap in<br />
solution the binding studies need to be enriched by measurements at different experimental<br />
conditions. This is a starting point to analyse possible intermolecular processes that can<br />
accompany binding <strong>of</strong> cap analogues by <strong>eIF4E</strong>: protonation or ion and water exchange.<br />
1.2.2. Gibbs Free Energy<br />
Chemical potential (μi) <strong>of</strong> interacting species at given conditions consists on the<br />
standard potential (μi°, index "°" refers to the pseudostandard state at concentrations <strong>of</strong> 1<br />
mol/dm 3 , i.e. unit molarity 127 ) and a contribution (μi m ) which depends on the presence <strong>of</strong><br />
other solution components (xi): 125<br />
o<br />
m<br />
μ = μ T,<br />
p)<br />
+ μ ( T,<br />
p,<br />
x ,..., x )<br />
Eq. 1-4<br />
i i ( i 1 α−1<br />
The activity <strong>of</strong> species is defined as:<br />
⎛ m<br />
⎞<br />
⎜<br />
μi<br />
( T,<br />
p,<br />
x1,...,<br />
x α−1<br />
)<br />
a<br />
≡<br />
⎟<br />
i ( T,<br />
p,<br />
x1,...,<br />
x α−1<br />
) exp<br />
, Eq. 1-5<br />
⎜<br />
⎟<br />
⎝<br />
RT<br />
⎠<br />
m<br />
so: i ≡ RT ln a i<br />
μ . Eq. 1-6<br />
14
Definition <strong>of</strong> the molar Gibbs free energy change (ΔG):<br />
∑ α<br />
i<br />
i=<br />
1<br />
ΔG<br />
≡ μ ν<br />
Eq. 1-7<br />
at equilibrium, where:<br />
m<br />
Δ ∑ i<br />
i 1<br />
α<br />
=<br />
i<br />
G = ΔG°<br />
+ μ νi<br />
= 0<br />
leads to the relationship for the standard molar Gibbs free energy change (ΔG°):<br />
o<br />
t<br />
15<br />
Eq. 1-8<br />
Δ G = −RT<br />
ln K . Eq. 1-9<br />
Stability <strong>of</strong> a noncovalent complex is determined by the difference in noncovalent<br />
interactions involving both the macromolecular recognition surfaces and solvent<br />
components in the complex and in the uncomplexed state. Endeavour to interpret the<br />
apparent Gibbs free energy <strong>of</strong> binding (ΔG°app) defined by the apparent association<br />
constant (Kas) and not by the thermodynamic association constant must take into account<br />
the fact <strong>of</strong> the participation <strong>of</strong> water, electrolyte ions and other solution components in the<br />
interactions <strong>of</strong> a protein <strong>with</strong> a ligand, as well as the other macromolecular equilibria.<br />
For shortening, the apparent Gibbs free energy change (ΔG°app) will be simply<br />
referred to here as "the Gibbs free energy change" but one should bear in mind that this<br />
"ΔG°" will be related to the observed and not to the thermodynamic association constant.<br />
1.2.3. <strong>Thermodynamic</strong> Parameters <strong>of</strong> Macromolecular <strong>Interaction</strong>s in<br />
Solution<br />
The Gibbs free energy change in an isothermal-isobaric process is related to changes<br />
in standard molar enthalpy (ΔH°) and entropy (ΔS°): 128<br />
ΔG ° = ΔH°<br />
− TΔS°<br />
. Eq. 1-10<br />
At constant but different temperatures the process is described by different pairs <strong>of</strong><br />
the ΔH° and ΔS° values, since they can vary <strong>with</strong> temperature according to the standard<br />
molar heat capacity change at constant pressure ( Δ ) and to two critical temperatures that<br />
are characteristic for the analysed process:<br />
o o<br />
H p H<br />
Δ = ΔC<br />
( T − T ) ,<br />
o<br />
C p<br />
o<br />
Δ H = 0 at TH; Eq. 1-11<br />
o o ⎛ T ⎞<br />
ΔS<br />
= ΔC<br />
p ln<br />
⎜<br />
⎟ , ΔS° = 0 at TS; Eq. 1-12<br />
⎝ TS<br />
⎠
⎛ ∂H°<br />
⎞<br />
C p = ⎜ ⎟<br />
⎝ ∂T<br />
⎠ p<br />
Δ o<br />
The parameters <strong>of</strong> interest can be determined from the van't H<strong>of</strong>f equation:<br />
ln K<br />
as<br />
o p<br />
ΔC<br />
⎡TH<br />
⎛ TS<br />
⎞ ⎤<br />
= ⎢ − ln⎜<br />
⎟ −1⎥<br />
R ⎣ T ⎝ T ⎠ ⎦<br />
16<br />
Eq. 1-13<br />
Eq. 1-14<br />
The isobaric heat capacity in solution is related to the fluctuations in internal energy<br />
(E) <strong>of</strong> the system ( X denotes an ensemble average <strong>of</strong> a quantity X):<br />
B<br />
( ) 2<br />
E E<br />
1<br />
C p ≈ C v = −<br />
Eq. 1-15<br />
2<br />
k T<br />
In most cases, the thermodynamic properties <strong>of</strong> the system expressed by ΔCp° are<br />
dominated by changes in water structure induced by macromolecular association. The<br />
increased energy fluctuations are related to lack <strong>of</strong> possibility <strong>of</strong> formation <strong>of</strong> hydrogen<br />
bonds by water molecules forced to contact <strong>with</strong> nonpolar (nonpolarizable) or aromatic<br />
(polarizable) solvent accessible groups <strong>of</strong> macromolecules (so called "hydrophobic<br />
effect"). The nature <strong>of</strong> aromatic rings shows that the terms "hydrophobic" and "nonpolar,<br />
apolar" must not be used equivalently, since the aromatic systems can carry induced charge<br />
distribution, being then partly polar, yet still hydrophobic objects. 129 Consequently, their<br />
contribution to the heat capacity changes, related to changes in the solvent accessible<br />
surface area (SASA), differs from that <strong>of</strong> aliphatic groups 130 (Table 1-3). The fluctuations<br />
<strong>of</strong> water energy are silenced when hydrogen bonds are maximally satisfied, i. e. when<br />
polar (uncharged) or charged molecular surface is water exposed. The second main origin<br />
<strong>of</strong> energy fluctuation in the system is related to macromolecular internal s<strong>of</strong>t vibrations. 131<br />
Table 1-3. Increments to the standard molar heat capacity resulting from exposure <strong>of</strong> 1 Å <strong>of</strong><br />
aliphatic, aromatic, and polar molecular surface to water (ΔCp°/SASA); data from different groups<br />
<strong>of</strong> authors.<br />
ΔCp° transfer /SASA (J⋅mol -1 ⋅K -1 ⋅Å -2 ) Authors (date) ref<br />
Surface: aliphatic aromatic polar<br />
+1.88 ± 0.08 a<br />
−1.09 ± 0.13 Murphy et al. (1991, 1992) 132,133<br />
+1.34 ± 0.17 a<br />
−0.59 ± 0.17 Spolar et al. (1992) 134<br />
+2.14 +1.55 −1.27 Makhatadze & Privalov (1995) 130<br />
a average values for aliphatic and aromatic surfaces
1.2.4. Noncovalent <strong>Interaction</strong>s in Aqueous Solutions<br />
"Energy <strong>of</strong> hydrogen bonds ranges from −12.5 to −38 kJ/mol" is the habitually<br />
repeated statement in the context <strong>of</strong> protein stability and protein-ligand interactions, both<br />
in many biochemical textbooks 135-137 as well as during academic lectures. However, this is<br />
true only if the "energy" means the internal energy (ΔE°) calculated usually in vacuum,<br />
and not the enthalpy (ΔH°) and Gibbs free energy (ΔG°). Binding between<br />
macromolecules in aqueous solution is an exchange, entropy-related process involving<br />
both water and solutes, since it needs disruption <strong>of</strong> pre-existing interactions <strong>of</strong> individual<br />
groups <strong>of</strong> each macromolecule <strong>with</strong> water and ions, and replacement <strong>of</strong> these interactions<br />
by contacts between complementary groups on each macromolecule. Hence, the stability<br />
<strong>of</strong> noncovalent complexes is totally different than in vacuum, and is highly dependent on<br />
the details <strong>of</strong> the solution environment. The real Gibbs free energy <strong>of</strong> the hydrogen bond<br />
formed in the water accessible region <strong>of</strong> the macromolecule dissolved in aqueous solution<br />
is only from −3 to −8 kJ/mol, 138-143 which may appear surprising but is a well documented<br />
rule. This is due mainly to entropic effects related to the presence <strong>of</strong> surrounding polar<br />
water molecules that compete for the hydrogen bond formation <strong>with</strong> the residues <strong>of</strong> the<br />
macromolecule. The strong decrease in free energy <strong>of</strong> a single amide hydrogen bond<br />
formation in water in comparison <strong>with</strong> that in apolar solvent (CCl4), from –35.31 ± 0.46 to<br />
−1.42 ± 1.2 kJ/mol, has been also demonstrated theoretically. 144 Similar entropic effects<br />
due to the presence <strong>of</strong> counterions should be taken into account when analysing the Gibbs<br />
free energy <strong>of</strong> salt bridges (Coulombic interactions <strong>with</strong> some contribution <strong>of</strong> hydrogen<br />
bonding) that usually link unlikely ionised residues.<br />
The second factor decreasing ΔG° <strong>of</strong> hydrogen bonds and salt bridges in<br />
macromolecules is the entropic penalty for ordering, e. g. conformational entropy <strong>of</strong><br />
protein side chains packing. Consequently, for macromolecules and their complexes<br />
interacting in buffered aqueous solutions (dielectric constant ε ~ 80, ionic strength I ~ 100-<br />
150 mM), the free energy <strong>of</strong> stabilization <strong>of</strong> a hydrogen bond is the same as that <strong>of</strong> a salt<br />
bridge, van der Waals interaction (induction and dispersion forces), and a hydrophobic<br />
contact. 145<br />
Stacking <strong>of</strong> aromatic rings (van der Waals and hydrophobic interactions) can attain<br />
free energy from 0 to −42 kJ/mol, depending on interatomic distances, but the energy is<br />
usually close to that <strong>of</strong> hydrogen bonds in proteins, 4 - 12.5 kJ/mol. 128,146,147 A nontypical<br />
17
ut biologically important example <strong>of</strong> stacking is cation - π stacking which consists in<br />
interaction between a charge and an induced quadrupole moment <strong>of</strong> charge distribution at<br />
the aromatic ring. 129,148,149<br />
Each type <strong>of</strong> noncovalent bonds has a typical range <strong>of</strong> interatomic distances. The<br />
optimal configuration <strong>of</strong> hydrogen bonds and salt bridges is linear, <strong>with</strong> the atoms/ions<br />
distant by 2.6 – 3.1 Å. The optimal distance for the van der Waals interaction is usually by<br />
0.3 - 0.5 Å greater than the sum <strong>of</strong> the van der Waals atomic radii, and this is ~ 4 Å for the<br />
C – C pair.<br />
1.2.5. Parsing <strong>of</strong> Gibbs Free Energy<br />
At issue is widely utilized parsing <strong>of</strong> ΔG° into contributions from individual,<br />
specific, non-covalent interactions, 120,137,139,150,151 i. e. hydrogen bonding, salt bridges, van<br />
der Waals contacts etc., from stating it to be meaningful 152 to proving its unreliability. 153<br />
The argument against parsing is based on the fact that even if the energy <strong>of</strong> the system can<br />
be approximated as linear combination <strong>of</strong> terms, it is not possible to write such general<br />
expression for the total free energy. The ΔG° value can be approximately factorized only<br />
when the resulting interactions are not correlated, e. g. operate on different coordinates.<br />
The free energy parsing gives reliable physical insight into macromolecular processes as<br />
long as the experimentally or theoretically obtained data are interpreted <strong>with</strong> care. 137,152,154<br />
1.2.6. <strong>Thermodynamic</strong>s <strong>of</strong> Coupled Processes<br />
<strong>Thermodynamic</strong>ally coupled processes that accompany binding <strong>of</strong> a ligand to a<br />
protein influence the thermodynamic parameters <strong>of</strong> binding to be determined. 122 In<br />
particular, the existence <strong>of</strong> many <strong>of</strong> protein conformational microstates can significantly<br />
shift the binding equilibrium in comparison <strong>with</strong> that for the binding to the single state.<br />
Additionally, conformational rearrangement <strong>of</strong> the ligand as well as protonation, ionic and<br />
hydration equilibria <strong>with</strong>in the interacting molecules can modulate the resultant<br />
thermodynamic features <strong>of</strong> the complex formation.<br />
A0<br />
1K, 1ΔH°<br />
(+B) K0, ΔH°0 K1, ΔH°1 (+B)<br />
BA0<br />
18<br />
A1<br />
A1B
For instance, if the molecule (A) exists in equilibrium <strong>of</strong> only two states (A0, A1) that<br />
is described by the constant (1K) and the transition enthalpy (1ΔH°), and can bind the<br />
second molecule (B) <strong>with</strong> different affinities and binding enthalpies <strong>of</strong> K0, K1 and ΔH°0,<br />
ΔH°1, respectively, then the observed parameters are as follows: 122<br />
( ΔH°<br />
+ ΔH°<br />
− ΔH°<br />
)<br />
1ΔH°⋅1<br />
K γ⋅1<br />
K 1<br />
1 0<br />
Δ H°<br />
= ΔH°<br />
0 − +<br />
; Eq. 1-16<br />
1+<br />
K<br />
1 + γ⋅<br />
K<br />
1<br />
( 1+<br />
K)<br />
1<br />
19<br />
( 1ΔH°<br />
+ ΔH°<br />
1 − ΔH°<br />
0 )<br />
T(<br />
1+<br />
γ⋅<br />
K)<br />
1ΔH°⋅1<br />
K<br />
γ⋅1<br />
K<br />
Δ S°<br />
= ΔS°<br />
0 − R ln(<br />
1+<br />
1K)<br />
− + R ln(<br />
1+<br />
γ⋅1<br />
K)<br />
+<br />
; Eq. 1-17<br />
T<br />
p<br />
p 0<br />
2 ( 1ΔH°<br />
) ⋅1<br />
2<br />
RT ( 1+<br />
K)<br />
1<br />
2<br />
1<br />
( )<br />
( ) 2<br />
1ΔH°<br />
+ ΔH°<br />
1 − ΔH°<br />
0<br />
2<br />
RT 1+<br />
γ⋅<br />
K<br />
K γ⋅1<br />
K<br />
Δ C°<br />
= ΔC°<br />
−<br />
+<br />
; Eq. 1.18<br />
where γ = K1/K0 (K1 < K0, γ [0,1]), and terms indexed "0" denote intrinsic parameters<br />
for binding <strong>of</strong> B to A in the A0 state. This case is called "nonmandatory coupling" since<br />
both A states can participate in the binding to some extent. In contrast, "mandatory<br />
coupling" occurs when only one state <strong>of</strong> A is capable <strong>of</strong> binding B, then K1 = 0 and γ = 0.<br />
In this case, the equations simplify significantly.<br />
Due to the two-state transition <strong>of</strong> A, the resultant binding free energy is always<br />
shifted toward less negative values:<br />
⎛ 1+<br />
⎞<br />
⎜ 1K<br />
ΔG°<br />
= ΔG°<br />
0 + RT ln<br />
⎟ . Eq. 1-19<br />
⎝1<br />
+ γ⋅1<br />
K ⎠<br />
It is interesting that macromolecular equilibria that have no intrinsic heat capacity<br />
changes resulting from surface-related phenomena and conformational stiffening (silencing<br />
<strong>of</strong> internal s<strong>of</strong>t modes) can yield a non-zero heat capacity change due only to the presence<br />
<strong>of</strong> coupled processes, e. g. due to ligand-induced shift in the equilibrium <strong>of</strong> protein states.<br />
The heat capacity change resulting from mandatory coupling is always negative, while the<br />
nonmandatory coupling can yield both negative and positive contribution, depending on<br />
the particular values <strong>of</strong> equilibrium constants and enthalpy changes.<br />
Hence, the overall heat capacity change <strong>of</strong> association that is accompanied by<br />
coupled processes can be written as a sum <strong>of</strong> the following terms:<br />
ΔC<br />
o p<br />
= ΔC<br />
+ ΔC<br />
o transfer<br />
p aliphatic<br />
o transfer<br />
+ ΔCp<br />
aromatic<br />
o<br />
p mandatory _ coupling<br />
+ Δ<br />
+ ΔC<br />
C<br />
o transfer<br />
p polar<br />
o<br />
p nonmandatory<br />
_ coupling<br />
1<br />
+ ΔC<br />
2<br />
o<br />
pint<br />
ernal _ s<strong>of</strong>t _ vibrations<br />
1<br />
+<br />
Eq. 1-20
2. Aim <strong>of</strong> The Work<br />
While the structure determinations provided important information on the mode <strong>of</strong><br />
cap binding, the consistent energetic description <strong>of</strong> the cap-<strong>eIF4E</strong> interaction was still<br />
lacking. In order to build a biophysical basis <strong>of</strong> the cap-dependent translation initiation and<br />
explain some discrepancies among structural, biochemical and biological<br />
observations, 75,155-157 measurements <strong>of</strong> molecular interactions in solution were necessary.<br />
The <strong>eIF4E</strong> protein possesses eight tryptophans which are conserved both in number<br />
and location, 21 and binding <strong>of</strong> cap results in quenching <strong>of</strong> the intrinsic protein<br />
fluorescence. 5,6,156,158-164 However, previous fluorescence measurements <strong>of</strong> the interaction<br />
between cap analogues and <strong>eIF4E</strong> purified by m 7 G-affinity chromatography yielded<br />
puzzling data. The reported association constants, 5,156,161-163 in the range <strong>of</strong> 10 5 -10 6 M -1 , did<br />
not reflect the differing inhibitory potency <strong>of</strong> cap analogues observed in vitro, 157 and<br />
contrasted <strong>with</strong> the structural data. 75,91 The crystal structure suggested that the binding<br />
constant for the <strong>eIF4E</strong>-m 7 GDP complex should be a few orders <strong>of</strong> magnitude higher, since<br />
there was a clearly visible hole in the middle <strong>of</strong> the six-membered ring <strong>of</strong> 7-<br />
methylguanosine in the electron density omit map. Such a high resolution <strong>with</strong>in the<br />
protein binding centre is usually observed by crystallographers for the complexes that have<br />
binding constants much higher than 10 6 . 165 These divergences were a prompt to perform<br />
systematic measurements to reveal the true affinity <strong>of</strong> cap analogues for <strong>eIF4E</strong>.<br />
The work was focused on the process <strong>of</strong> association <strong>of</strong> recombinant untagged murine<br />
<strong>eIF4E</strong> (residues 28-217) and full length human <strong>eIF4E</strong> <strong>with</strong> a large series <strong>of</strong> mono- and<br />
dinucleotide cap analogues, in order to attain several particular goals leading together to a<br />
complete decription <strong>of</strong> the molecular mechanism <strong>of</strong> specific recognition <strong>of</strong> the <strong>mRNA</strong> 5'<br />
structure by <strong>eIF4E</strong>. The particular goals were as follows:<br />
1. determination <strong>of</strong> association constants that would reflect the structure-activity<br />
relationship;<br />
2. parsing <strong>of</strong> the free energy <strong>of</strong> the binding into contributions <strong>of</strong> individual structural<br />
elements in the cap-binding centre,<br />
3. elucidation <strong>of</strong> the nature <strong>of</strong> the stacking-hydrogen bonding cooperativity,<br />
4. searching for conformational changes and water exchange between the complex and<br />
bulk solution,<br />
20
5. revealing the role for ionic equilibria and electrostatic interactions in cap-<strong>eIF4E</strong><br />
recognition,<br />
6. analysis <strong>of</strong> the specific binding between <strong>eIF4E</strong> and <strong>mRNA</strong> 5' cap in terms <strong>of</strong> exact<br />
quantitative thermodynamic parameters determined independently from the van't<br />
H<strong>of</strong>f equation and isothermal titration calorimetry (ITC).<br />
Such quantitative data are <strong>of</strong> primary importance for rational design <strong>of</strong> new cap-<br />
analogues <strong>of</strong> potential therapeutic activity since the high <strong>eIF4E</strong> cellular level is relevant to<br />
malignancy and apoptosis 13 .<br />
The fluorescence affinity measurements have been extended to include ternary<br />
complexes which consisted <strong>of</strong> <strong>eIF4E</strong>, a cap-analogue, and synthetic peptides corresponding<br />
to the <strong>eIF4E</strong> recognition motifs from the mammalian proteins eIF4GI, eIF4GII, 4E-BP1,<br />
and 4E-BP1 peptides monophosphorylated at Ser65 and diphosphorylated at Ser65/Thr70.<br />
The particular questions asked in the work were related to two issues:<br />
1. what is the influence <strong>of</strong> phosphorylation at Ser65 and Thr70 on regulation <strong>of</strong> 4E-BP1<br />
binding to <strong>eIF4E</strong>,<br />
2. how to reconcile a lot <strong>of</strong> contradictory biological and biochemical experimental data<br />
reported hitherto, regarding the putative cooperation between two <strong>eIF4E</strong> binding<br />
sites. 166-168<br />
21
3. Materials and Methods<br />
3.1. Chemical and Biochemical Syntheses<br />
3.1.1. Cap Analogues<br />
Syntheses and purification <strong>of</strong> cap analogues (Scheme 3-1) as sodium salts were<br />
performed by Dr. Dr. Edward Dar¿ynkiewicz, Janusz Stêpiñski and Marzena Jankowska-<br />
Anyszka as described previously 169-173 . Their concentrations were obtained from weighed<br />
amounts (± 5 %) and checked spectrophotometrically. 157 The cap analogues in solution at<br />
elevated pH (> 8) undergo opening <strong>of</strong> the five-membered ring <strong>of</strong> 7-substituted guanine,<br />
followed by hydrolysis <strong>of</strong> the glycosidic bond. 174 As checked by NMR, 175 the cap was very<br />
stable at pH 7.2, and stable enough to perform fast experiments at higher pH.<br />
3.1.2. Peptides<br />
Peptides corresponding to residues 569-580 <strong>of</strong> mammalian eIF4GI:<br />
KKRYDREFLLGF, 34 621-637 <strong>of</strong> mammalian eIF4GII: KKQYDREFLLDFQFMPA, 35 and<br />
51-67 <strong>of</strong> mammalian 4E-BP1: RIIYDRKFLMECRNSPV 67 were synthesized by Dr. Joseph<br />
Marcotrigiano (The Rockefeller University, N.Y., U.S.A.) by Boc protocols for solid phase<br />
peptide synthesis and cleaved using a standard HF procedure. 176 Syntheses <strong>of</strong> a<br />
mammalian phosphopeptide P-Ser65 51-67 4E-BP1 and a diphosphopeptide P-<br />
Ser65/Thr70 51-75 4E-BP1 RIIYDRKFLMECRNSpPVTKTpPPKDL 67 were performed<br />
by Dr. Aleksandra Wys³ouch-Cieszyñska (IBB PAS, Warszawa) on a Wang resin<br />
(Novabiochem) using Fmoc protocols for solid phase phosphopeptide synthesis. 1 Peptides<br />
were purified to homogeneity by semi-preparative HPLC (> 95 % purity), and<br />
characterized by MALDI-TOF or ESI mass spectrometry (569-580 eIF4GI peptide,<br />
predicted mass 1697.0 Da, measured mass 1698.0 Da; 621-637 eIF4GII peptide, predicted<br />
mass 2177.8 Da, measured mass 2178.0 Da; 51-67 4E-BP1 peptide, predicted mass 2141.8<br />
measured mass 2141; phosphopeptide, P-Ser65 51-67 4E-BP1, predicted mass 2220.1,<br />
measured mass 2221.0; diphosphopeptide, P-Ser65/Thr70 51-75 4E-BP1, predicted mass<br />
3180.55, measured mass 3181.0).<br />
Concentrations <strong>of</strong> the five investigated peptides were determined by acid gas-phase<br />
hydrolysis and three independent repeats <strong>of</strong> amino acid analysis, by Dr. Pawe³ Mak<br />
(Jagiellonian University, Kraków).<br />
22
Scheme 3-1. Structures <strong>of</strong> the 16 methylated cap-analogues. Φ denotes the phenyl ring. Protons<br />
which partially dissociate at pH 7.2, are marked <strong>with</strong> asterisk (pKa N(1)-H ~7.24-7.54, depending on<br />
R2, R3, and n 177 ; pKa phosph ~6.1-6.5, depending on n 178 ).<br />
m 7 GTP: R1 = CH3, R2 = R3 = H, n = 3;<br />
m 7 GDP: R1 = CH3, R2 = R3 = H, n = 2;<br />
m 7 GMP: R1 = CH3, R2 = R3 = H, n = 1;<br />
m 7 G: R1 = CH3, R2 = R3 = H, n = 0;<br />
et 7 GTP: R1 = CH2-CH3, R2 = R3 = H, n = 3;<br />
bz 7 GTP R1 = CH2-Φ, R2 = R3 = H, n = 3;<br />
p-Cl-bz 7 GTP: R1 = CH2-Φ-Cl, R2 = R3 = H, n = 3;<br />
m 7 , 2<br />
2 GTP : R1 = CH3, R2 = CH3, R3 = H, n = 3;<br />
m 7 , 2 , 2<br />
R2<br />
R2<br />
* H<br />
N<br />
R3<br />
*<br />
H<br />
N<br />
R3<br />
O<br />
N 6<br />
1<br />
2<br />
3<br />
N<br />
N<br />
O<br />
N<br />
R1<br />
N<br />
7<br />
+<br />
9<br />
N<br />
8<br />
H<br />
H<br />
H<br />
H<br />
R1<br />
N<br />
+<br />
N<br />
H<br />
OH<br />
HO<br />
3 GTP : R1 = CH3, R2 = R3 = CH3, n = 3;<br />
O<br />
H<br />
H H<br />
OH<br />
HO<br />
O<br />
m 7 Gpppm 2’O G: R1 = CH3, R2 = R3 = H, n = 3, R4 = OCH3, B = G<br />
m 7 GpppG: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = G<br />
m 7 GpppA: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = A<br />
m 7 GpppC R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = C<br />
m 7 Gpppm 7 G: R1 = CH3, R2 = R3 = H, n = 3, R4 = OH, B = m 7 G<br />
m 7 GppppG: R1 = CH3, R2 = R3 = H, n = 4, R4 = OH, B = G<br />
m 7 Gppppm 7 G: R1 = CH3, R2 = R3 = H, n = 4, R4 = OH, B = m 7 G<br />
23<br />
CH 2<br />
H<br />
CH 2<br />
H<br />
O<br />
O (P O) n<br />
O -<br />
O<br />
O (P O) n CH 2<br />
O -<br />
H<br />
H *<br />
H<br />
OH<br />
O<br />
H<br />
B<br />
H<br />
R4
A dodecapeptide DGIEPMWEDEKN was kindly provided by Dr. Laslo Balaspiri<br />
(A. Szent-Gyorgyi Medical University, Szeged, Hungary).<br />
All other chemicals were analytical grade, purchased from Sigma-Aldrich, Merck,<br />
Carl Roth (Germany) or Fluka (U.S.A.).<br />
3.1.3. <strong>Protein</strong> Biosynthesis<br />
Human eukaryotic initiation factor <strong>eIF4E</strong> (residues 1-217) and murine <strong>eIF4E</strong><br />
(residues 28-217 and residues 33-217) was expressed by Mrs. Lidia Chlebicka <strong>with</strong> a help<br />
<strong>of</strong> Dr. Micha³ Dadlez (IBB PAS, Warszawa) in Escherichia coli (strain<br />
BL21(DE3)pLys). 179,180 The bacterial cells were transformed by a pET11d plasmid,<br />
containing the cloned coding region for <strong>eIF4E</strong>, and the T4 promoter. Induction <strong>of</strong> T7<br />
polymerase in liquid culture <strong>of</strong> bacteria on Luria-Bertani broth, <strong>with</strong> ampicillin and<br />
chloramphenicol, was initiated by addition <strong>of</strong> isopropyl-β-D-galactopyranoside. Bacterial<br />
pellets were lysed by sonication.<br />
The human protein was initially isolated from the soluble fraction by means <strong>of</strong><br />
affinity chromatography 181 and purified on an ion exchange MonoQ column. Next, the<br />
human and the murine proteins were purified from inclusion bodies pellets and folded by<br />
one step dialysis from 6 M guanidine hydrochloride, followed by ion-exchange<br />
chromatography on a MonoQ (human) or MonoS (murine) column, thus avoiding contact<br />
<strong>with</strong> cap analogues at any stage <strong>of</strong> purification. The protein purity, checked by a routine<br />
SDS PAGE, was > 95 %.<br />
3.2. Preparation <strong>of</strong> <strong>Protein</strong> and Peptide Samples to<br />
Spectroscopic Measurements<br />
The protein solutions were buffer exchanged if necessary <strong>with</strong> use <strong>of</strong> Ultrafree-15 ml<br />
filters <strong>with</strong> Biomax 5 kDa NMWL membrane (Millipore Co., U.S.A.). Immediately before<br />
the spectroscopic measurements, the protein sample was filtered through Millipore<br />
Ultrafree-0.5 ml Biomax 100 kDa NMWL. Total concentration <strong>of</strong> <strong>eIF4E</strong> was determined<br />
from absorbance ( 280 53900 = ε cm-1M -1 ).<br />
Solutions <strong>of</strong> 4E-BP1 peptides were strictly controlled for lack <strong>of</strong> disulphide dimer<br />
formation. Dimers occurred in the 4E-BP1 solution because <strong>of</strong> lack <strong>of</strong> any additives in the<br />
stock water solutions, which was necessary for accurate determination <strong>of</strong> the peptide<br />
24
concentrations by amino acid analysis. Peptide dimers were broken if incubated at very<br />
high concentration <strong>of</strong> the reducing agent (13 mM DTT) at pH 7.5 for two weeks. This<br />
secured satisfactory monomerization, as rigorously checked on analytical HPLC (Waters<br />
Inc.) and ESI mass spectroscopy (Q-T<strong>of</strong> 2 spectrometer, Micromass Inc.). Then, the<br />
affinity measurements could be performed properly <strong>with</strong> use <strong>of</strong> more dilute peptide<br />
solution at 1 mM DTT. The problem concerned the phosphorylated 4E-BP1 peptides to<br />
lesser extent, since the presence <strong>of</strong> the negatively charged phosphate groups at Ser65 in the<br />
vicinity <strong>of</strong> Cys62 can partially prevent the dimerization.<br />
3.3. Absorption and Emission Spectroscopy<br />
Emission <strong>of</strong> light from a fluorophore occurs from electronically excited states. 182<br />
Return to the ground electronic state (S0) from excited singlet states (Sn) is attained by a<br />
rapid (after 10 -10 - 10 -8 s), spin-allowed emission <strong>of</strong> a photon (fluorescence), while<br />
transition from excited tripled states (phosphorescence) is forbidden and the lifetimes are<br />
longer (10 -3 – 10 1 s). Phosphorescence is not visible in aqueous solutions at temperatures<br />
applied during this work and will not be considered.<br />
Light absorption in UV-Vis wave region occurs typically from the lowest vibrational<br />
level (<strong>of</strong> S0) to some higher vibrational level <strong>of</strong> S1 or S2 in a time <strong>of</strong> about 10 -15 s. In<br />
solutions, molecules rapidly relax to the lowest vibrational level <strong>of</strong> S1 via internal<br />
conversion in a time ≤ 10 -12 s. Fluorescence return to S0 occurs to one <strong>of</strong> its vibrational<br />
levels. Then the molecule quickly reaches thermal equilibrium (10 -12 s). The rapid<br />
irradiative decay to the lowest vibrational level <strong>of</strong> S1 results in the Stokes' shift between the<br />
absorption and emission spectra. The shift is further enhanced by possible decay to higher<br />
vibrational levels <strong>of</strong> S0, reorientation <strong>of</strong> fluorophore in respect to solvent molecules,<br />
excited-state reactions, complex formation, and energy transfer.<br />
When incident light (intensity I0) is absorbed by a diluted chromophore solution<br />
(concentration [ch], extinction coefficient ε) on the optical length (l), then the absorbance<br />
(A) and the intensity <strong>of</strong> transmitted light (I1) can be reliably expressed by the linear Beer-<br />
Lambert law: 183<br />
light:<br />
I0<br />
A = log = ε ⋅[<br />
ch]<br />
⋅ l . Eq. 3-1<br />
I<br />
1<br />
The rate <strong>of</strong> emission <strong>of</strong> fluorescence (F) is related to the intensity <strong>of</strong> the absorbed<br />
25
F = (I0 − I1) ⋅ q, Eq. 3-2<br />
where q is the quantum yield <strong>of</strong> fluorescence. For weakly absorbing solutions for which<br />
the optical density is very small, the equation can be simplified to:<br />
F = 2.3⋅I0⋅ε⋅[ch]⋅l⋅q. Eq. 3-3<br />
Fluorescence intensities are proportional to the fluorophore concentration over a very<br />
limited range <strong>of</strong> optical densities, usually 2 orders <strong>of</strong> magnitude lower than those which<br />
satisfy the Beer-Lambert law for absorption. If the conditions ensuring linearity are<br />
satisfied, the observed fluorescence changes can be directly interpreted in terms <strong>of</strong><br />
changing concentrations <strong>of</strong> the species present in a studied sample. Otherwise suitable<br />
corrections for the inner filter effect are necessary 183 (see below, 3.4.2.1., p. 31).<br />
3.3.1. Fluorescence Quenching<br />
Fluorescence intensity can be decreased by: 182<br />
1. dynamic quenching:<br />
a) irradiative return do S0 by a diffusive collisions <strong>of</strong> the excited fluorophore <strong>with</strong><br />
other molecules,<br />
b) resonance energy transfer (RET) <strong>of</strong> the excited-state energy from an excited donor<br />
to an acceptor <strong>with</strong>out the appearance <strong>of</strong> a photon, as a result <strong>of</strong> long-range<br />
dipole-dipole interactions,<br />
2. static quenching by formation <strong>of</strong> nonfluorescent complexes <strong>of</strong> fluorophore <strong>with</strong> a<br />
quencher in the fluorophore ground state,<br />
3. inner filter effect, i. e. attenuation <strong>of</strong> the excitation beam and/or reabsorption <strong>of</strong> the<br />
emitted beam by a layer <strong>of</strong> an optically dense solution.<br />
3.3.1.1. Static Quenching<br />
Static quenching results from formation <strong>of</strong> a dark complex that returns to the ground<br />
state immediately after absorption <strong>with</strong>out emission <strong>of</strong> a photon. A fraction <strong>of</strong> the<br />
fluorophores is removed from observation and, contrary to dynamic quenching, the<br />
lifetime <strong>of</strong> the uncomplexed fraction is unchanged. The quenching constant is the<br />
association constant <strong>of</strong> the dark complex: 182<br />
[ cx]<br />
K s = , Eq. 3-4<br />
[ f ] ⋅[<br />
qu]<br />
0<br />
0<br />
26
where [cx], [f]0 and [qu]0 are the equilibrium concentrations <strong>of</strong> the complex, free<br />
fluorophore and free quencher, respectively. The fluorescence changes analogically as<br />
upon the dynamic quenching (below):<br />
F0<br />
= 1+<br />
K s[<br />
qu]<br />
0 , Eq. 3-5<br />
F<br />
but the equilibrium concentration the free quencher must be analysed here.<br />
3.3.1.2. Dynamic Quenching<br />
Dynamic quenching by a quencher at a total concentration [qu] is a rate process<br />
which depopulates the excited state, leading to shortening <strong>of</strong> fluorescence lifetimes (τ0).<br />
The resultant lifetime in the presence <strong>of</strong> the quencher (τ) is shorter by a decay rate (kq)<br />
which is characteristic to the given quencher and environment:<br />
τ0<br />
= 1+ k qτ0<br />
[ qu]<br />
. Eq. 3-6<br />
τ<br />
An equivalent decrease in fluorescence intensity is:<br />
0 F<br />
F<br />
τ<br />
=<br />
τ<br />
0<br />
. Eq. 3-7<br />
The Stern-Volmer quenching constant is defined as:<br />
KSV = kq ⋅ τ0, Eq. 3-8<br />
so that the Stern-Volmer equation is expressed as:<br />
F0<br />
= 1+<br />
K SV[<br />
qu]<br />
. Eq. 3-9<br />
F<br />
3.3.1.3. Resonance Energy Transfer<br />
The rate <strong>of</strong> RET (kT) is related to a factor describing the relative space orientation <strong>of</strong><br />
the transition dipoles <strong>of</strong> the donor and acceptor (κ 2 ), to the quantum yield <strong>of</strong> the donor (qd),<br />
to the lifetime <strong>of</strong> the donor in the absence <strong>of</strong> the acceptor (τd), to the reciprocal <strong>of</strong> sixth<br />
power <strong>of</strong> their distance (r), and to the integral <strong>of</strong> spectral overlap between the donor<br />
emission and the acceptor absorption (J(λ)):<br />
k<br />
T<br />
q<br />
~<br />
τ<br />
d<br />
d<br />
⋅ κ<br />
⋅ r<br />
2<br />
6<br />
J(<br />
λ)<br />
. Eq. 3-10<br />
27
The distance at which the transfer rate is equal to the decay rate <strong>of</strong> the donor in the<br />
absence <strong>of</strong> acceptor (by the radiative and nonradiative ways) is called the Förster distance<br />
(R0), usually ~ 30 – 60 Å.<br />
The orientation factor (κ 2 ) is given by:<br />
2<br />
κ = cos θ − 3cos<br />
θ ⋅ cos θ , Eq. 3-11<br />
T<br />
d<br />
a<br />
where θT is the angle between the donor and acceptor dipoles, θd and θa are the angles<br />
between these dipoles and the vector joining them. The κ 2 value can range from 0 for the<br />
perpendicular orientation <strong>of</strong> the dipoles to 4 for the collinear, parallel orientation.<br />
3.3.1.3.1. Resonance Energy Transfer in <strong>eIF4E</strong><br />
The <strong>eIF4E</strong> molecule contains 6 tyrosines and 8 tryptophans. 50 The diameter <strong>of</strong> the<br />
molecule is ~ 40 Å, i. e. in the typical range <strong>of</strong> the Förster distances. Hence, the tyrosine<br />
(donor) fluorescence can be transferred to tryptophans (acceptor), 182 and the shape <strong>of</strong> the<br />
emission spectrum excited at 280 nm is dominated by the final tryptophan fluorescence<br />
(Fig. 4-1, p. 49). It has been checked by comparison <strong>of</strong> <strong>eIF4E</strong> emission spectra registered<br />
for different excitation wavelengths (λex) that the contribution <strong>of</strong> the tyrosine emission in<br />
the resultant fluorescence intensity at λex = 280 nm is not more than 10 %. Putative<br />
conformational changes can modulate both tyrosine and tryptophan emission but<br />
eventually the spectral information about the changes is provided by the tryptophan<br />
emission.<br />
One issue is the interaction <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the tyrosine containing peptides 4E-BP1,<br />
4E-BP1 P-Ser65, 4E-BP1 P-Ser65/Thr70, eIF4GI, and eIF4GII. The peptide tyrosine is<br />
distant by only 10 Å from Trp73 <strong>of</strong> <strong>eIF4E</strong> in the bound state. 91 However, the mutual<br />
orientation <strong>of</strong> the two aromatic rings is almost perpendicular (Fig. 4-22, 23, p. 86-87). The<br />
tyrosine is stabilized in this plane by a close phenylalanine ring and an –OH hydrogen<br />
bond, and the tryptophan is tightly bound by a network <strong>of</strong> intra- and intermolecular<br />
interactions. These constraints suggest that in spite <strong>of</strong> proximity <strong>of</strong> the donor and acceptor<br />
the RET is most probably inefficient.<br />
3.3.2. Tryptophan Fluorescence in <strong>Protein</strong>s<br />
Tryptophan fluorescence varies widely in quantum yield and in the wavelength <strong>of</strong><br />
maximum intensity among proteins and solvents. 182 The 280-nm absorption band <strong>of</strong> Trp is<br />
28
the result <strong>of</strong> transition to two excited states, called 1 La and 1 Lb, <strong>of</strong> considerably different<br />
properties. 184 The 1 La state has a large dipole moment, and thus the high sensitivity to<br />
environment. Transition from 1 La usually yields more fluorescence but 1 Lb can be<br />
sometimes also the emitting state and then they are hardly distinguishable in proteins. The<br />
transitions have different structures, intensities and relative spectral positions, but the<br />
details are blurred by inhomogeneous broadening in proteins due mainly to the variation <strong>of</strong><br />
the relative orientation <strong>of</strong> the local electric field felt by tryptophans. This solvent-solute<br />
interaction accounts for the large Stokes' shift in water and much less inside the protein<br />
hydrophobic core. The large red shift is related to the cooperative effect <strong>of</strong> many water<br />
molecules, ~ 100 cm -1 /1 water molecule <strong>with</strong>in 6 Å. 184 Additionally, protein tryptophans<br />
can be dynamically quenched by neighbouring charged amino acids. 182 Hence, the<br />
spectroscopic properties (quantum yield, accurate shape <strong>of</strong> the spectrum e.t.c.) <strong>of</strong> <strong>eIF4E</strong><br />
were not the main focus <strong>of</strong> the work to avoid too far-reaching, uncertain conclusions.<br />
Instead, the fluorescence intensity was used to monitor quantitatively the changes<br />
experienced by <strong>eIF4E</strong>.<br />
3.3.3. Quenching <strong>of</strong> <strong>eIF4E</strong> Fluorescence by Cap Analogues<br />
The intrinsic protein fluorescence quenching observed upon formation <strong>of</strong> the<br />
complex <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> a cap analogue can result from two factors: direct quenching <strong>of</strong> the<br />
tryptophans 102, 56 and 166 in the cap-binding site, and changes <strong>of</strong> local environment <strong>of</strong><br />
other, more distant fluorescent amino acids as a result <strong>of</strong> possible conformational changes<br />
<strong>of</strong> the protein upon the binding. The former is thought to be static quenching, since:<br />
1. The Förster resonance energy transfer in unlikely due to several reasons:<br />
a) Spectral overlapping <strong>of</strong> the cap analogue emission and the protein absorption<br />
spectra, and reversely (Fig. 4-1, p. 49), the protein emission and the cap analogue<br />
absorption spectra, is negligible.<br />
b) Neither any batochromically shifted emission band nor fluorescence<br />
enhancement <strong>of</strong> cap analogues were observed. The emission <strong>of</strong> the free cap in the<br />
presence <strong>of</strong> the protein obtained by fitting <strong>of</strong> the full equation (Eq. 3-26, p. 34)<br />
was the same as the emission in the absence <strong>of</strong> the protein;<br />
c) Brownian dynamic calculations involving electrostatic effects together <strong>with</strong> the<br />
stopped-flow binding studies 4 suggested that quenching can occur only when the<br />
ligand is apart from its final position known from the crystal structure 75 not more<br />
29
than a few angströms. It would probably still diminish this distance if polarization<br />
effects were taken into account in the calculations. 185<br />
2. Collisional quenching <strong>of</strong> the protein tryptophans seems to be unlikely at such low<br />
concentration <strong>of</strong> the protein, 50 nM – 1 μM, and <strong>of</strong> the cap analogue, not exceeding<br />
5 μM for the most specific analogue and 150 μM for the least specific one.<br />
However, the unambiguous assessment could be only provided by time-resolved<br />
measurements.<br />
Fluorescence changes <strong>of</strong> tyrosines, and the tryptophans which do not have direct<br />
contact <strong>with</strong> cap are related to dynamic quenching by surrounding charged amino acids, 182<br />
changes <strong>of</strong> mutual distances and orientations in Trp-Tyr pairs, and changes <strong>of</strong> local<br />
polar/hydrophobic neighbourhood. The quenching has a dynamic character but reflects the<br />
changes that the protein undergoes upon cap binding and thus depends not on the total<br />
quencher concentration but on the equilibrium concentration. Hence, it is described by the<br />
same equation as static quenching.<br />
3.4. Experimental Conditions <strong>of</strong> Spectroscopic Measurements<br />
Absorption and fluorescence spectra were recorded on Lambda 20 UV/VIS and LS-<br />
50B instruments (Perkin-Elmer Co., Norwalk, CT., USA), in a quartz semi-micro cuvette<br />
(Hellma, Germany) <strong>with</strong> optical lengths 4 mm and 10 mm for absorption and emission,<br />
respectively. The titration experiments were performed in a standard buffer: 50mM<br />
Hepes/KOH pH 7.20, 100mM KCl, 1mM dithiothreitol (DTT) and 0.5mM disodium<br />
ethylenediaminetetraacetate (EDTA), except for the experiments at variable pH and ionic<br />
strength. pH (± 0.01 unit) was measured independently at each temperature and ionic<br />
strength (Beckman Φ300 pH-meter, Germany). Solutions were filtered through 0.22 μm<br />
pore size. <strong>Protein</strong> samples were s<strong>of</strong>tly degassed prior to titration. The cuvette was<br />
thermostated and the temperature was controlled <strong>with</strong> a thermocouple inside it (± 0.2 °C).<br />
For the <strong>eIF4E</strong>-cap association the excitation wavelength <strong>of</strong> 280nm (slit 2.5nm, auto<br />
cut-<strong>of</strong>f filter), and the emission wavelength <strong>of</strong> 335, 336 or 337nm (slit 2.5 to 4nm, 290 nm<br />
cut-<strong>of</strong>f filter) were applied, <strong>with</strong> an automatic correction for the photomultiplier sensitivity.<br />
For the <strong>eIF4E</strong>-peptide binding studies, the excitation wavelength <strong>of</strong> 290nm and the<br />
emission wavelength <strong>of</strong> 350 or 355 nm were used. These conditions ensured observation <strong>of</strong><br />
the protein tryptophan emission, only. The fluorescence intensity was monitored by the<br />
30
egistration <strong>of</strong> the whole spectrum (310 nm to 400 nm) and during continuous, time-<br />
synchronized titration at a single wavelength, <strong>with</strong> the integration time <strong>of</strong> 30 seconds and<br />
the gap <strong>of</strong> 30 seconds for adding the ligand, <strong>with</strong> slow but sufficient magnetic stirring to<br />
ensure mixing and keeping the temperature constant in the whole volume <strong>of</strong> the cuvette.<br />
During the gap, the UV xenon flash lamp was switched <strong>of</strong>f to avoid photobleaching the<br />
photosensitive protein sample. The cuvette has not been touched during the whole titration<br />
experiment to ensure constant geometry for the optical measurements.<br />
3.4.1. Titration Assay<br />
Titrations were performed for <strong>eIF4E</strong> at several concentrations (50 nM to 1 μM), in<br />
steady-state conditions provided by preincubation <strong>of</strong> <strong>eIF4E</strong> in the buffer <strong>of</strong> the appropriate<br />
pH and ionic strength for the given experiment. 1 μl aliquots <strong>of</strong> increasing concentrations<br />
(1 μM to 5 mM) <strong>of</strong> a ligand were injected manually to 1400 μl <strong>of</strong> <strong>eIF4E</strong> solution. Each<br />
titration consisted <strong>of</strong> 28 to 45 data points <strong>with</strong> a suitable number <strong>with</strong>in the range, at which<br />
the total ligand concentration ([L]) was close to the concentration <strong>of</strong> active <strong>eIF4E</strong> ([Pact])<br />
(Fig. 4-7, p. 57). The curvature <strong>of</strong> the fitted function (Eq. 3-26, p. 34) in this range,<br />
∂<br />
2<br />
F<br />
∂[<br />
L]<br />
2<br />
[L] = [Pact<br />
]<br />
~<br />
K<br />
P<br />
as<br />
act<br />
, Eq. 3-12<br />
mostly influences the accuracy <strong>of</strong> fitting <strong>of</strong> [Pact] and Kas. For determination <strong>of</strong> the active<br />
protein fraction in the samples studied by stopped-flow fluorimetry and ITC, the cap<br />
analogue <strong>of</strong> the highest association constant for <strong>eIF4E</strong> was used (7-methylGTP), since this<br />
Kas ensures the optimal quotient <strong>of</strong> Kas/[Pact].<br />
3.4.2. Fluorescence Data Corrections<br />
3.4.2.1. Inner Filter Effect<br />
The fluorescence intensities were corrected for the inner filter effect 183 (Fig. 3-1).<br />
The cuvette had two thicker walls and thus the optical pathlength for absorption was<br />
significantly shorter than that for emission (4 mm × 10 mm), which minimized both the<br />
inner filter effect and a recapture <strong>of</strong> the excitation beam from a little mirror behind the<br />
thick cuvette wall. A following polynomial dependence <strong>of</strong> the fluorescence intensity (F) on<br />
the absorbance (A):<br />
F(<br />
A)<br />
2<br />
3<br />
4<br />
= 1−<br />
2.<br />
348⋅<br />
A + 7.<br />
56 ⋅ A − 20.<br />
2 ⋅ A + 21.<br />
5 ⋅ A<br />
Eq. 3-13<br />
F(<br />
0)<br />
31
proved to be better than the correction proposed for the typical 10 mm × 10 mm cuvette 186<br />
(P < 0.0001). The experimental data (Fobs) were divided by the above factor (F(A)/F(0))<br />
determined individually for each titration, yielding the data-set corrected for absorption<br />
(FcorrA):<br />
F(<br />
A)<br />
= F<br />
Eq. 3-14<br />
F(<br />
0)<br />
FcorrA obs<br />
The inner filter effect was negligible for the specific cap analogues but could change Kas<br />
values ~ 2-fold for the weakly interacting and strongly absorbing cap analogues (Fig. 3-1).<br />
F(A)/F(0)<br />
Residuals<br />
1.0<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.02<br />
0<br />
-0.02<br />
Figure 3-1. (left) Inner filter effect measured for tryptophan solution in the 4 mm × 10 mm cuvette,<br />
titrated by GMP in 50mM Hepes/KOH buffer, pH 7.20, 100mM KCl. A comparison <strong>of</strong> a<br />
polinomial function (solid line, black residuals) <strong>with</strong> the analytical function proposed for the 10<br />
mm × 10 mm cuvette 186 (broken line, open residuals). The maximal GMP concentration is < 100<br />
μM, which rules out a possibility <strong>of</strong> dynamic quenching <strong>of</strong> tryptophan. (right) Fluorescence<br />
titration <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> GDP. Data uncorrected () and corrected () for the inner filter effect.<br />
3.4.2.2. Dilution<br />
0.0 0.1 0.2 0.3 0.4<br />
Absorbance<br />
0.0 0.1 0.2 0.3 0.4<br />
The final dilution was always ≤ 3.2%. Suitable data correction was applied for these<br />
titrations for which the final dilution was ≥ 2%. FcorrV is the corrected fluorescence, Fobs is<br />
the measured fluorescence, V is the actual sample volume at each point <strong>of</strong> the titration, and<br />
V0 is the initial sample volume:<br />
V<br />
FcorrV = Fobs<br />
⋅ . Eq. 3-15<br />
V<br />
0<br />
Fluorescence (a. u.)<br />
32<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0 20 40 60 80 100 120<br />
GDP (μM)
3.4.2.3. Fluorescence Drift in Time<br />
In some cases at higher temperatures (~ 40 °C) the protein fluorescence decreased<br />
somewhat in time due most probably to partial thermal denaturation <strong>of</strong> <strong>eIF4E</strong>. A correction<br />
was needed to cancel numerically the fluorescence changes that did not result from ligand<br />
binding. Thanks to synchronization <strong>of</strong> each injection during titration, a correction for a<br />
fluorescence decrease in the time <strong>of</strong> the titration experiment could be applied. Monitoring<br />
<strong>of</strong> the fluorescence signal was continued after completing the titration by the next 10 - 20<br />
minutes in the same way as during the titration. The exponential curve was fitted to the<br />
time-dependent data at the constant ligand concentration:<br />
F ( t)<br />
= a ⋅ exp( −b<br />
⋅ t)<br />
+ c . Eq. 3-16<br />
The titration data were then corrected (FcorrT) by the exponential function:<br />
FcorrT obs<br />
= F ⋅ exp( b ⋅ t)<br />
. Eq. 3-17<br />
The latter two corrections had only slight influence on the resultant Kas values<br />
(<strong>with</strong>in 0.3 standard deviation) but improved the goodness <strong>of</strong> fit (R 2 and P value, see 3.9.,<br />
p. 46)<br />
3.5. Fluorescence Data <strong>Analysis</strong><br />
3.5.1. Simultaneous Determination <strong>of</strong> Association Constants and <strong>Protein</strong><br />
Activity<br />
As a result <strong>of</strong> binding <strong>of</strong> a ligand at a total ligand concentration <strong>of</strong> [L] to an active<br />
protein at a total concentration <strong>of</strong> [Pact], there are three species in the solution: the free<br />
ligand at the equilibrium concentration <strong>of</strong> [L]0, the free active protein at the equilibrium<br />
concentration <strong>of</strong> [Pact]0, and their complex at the equilibrium concentration <strong>of</strong> [cx]:<br />
[L] = [L]0 + [cx], Eq. 3-18<br />
[Pact] = [Pact]0 + [cx]. Eq. 3-19<br />
Equilibrium among the concentrations <strong>of</strong> free species and the complex is ruled by the<br />
association constant Kas that is defined in the same manner as the static quenching constant<br />
(Eq. 3-4):<br />
[ cx]<br />
K as = . Eq. 3-20<br />
[ L]<br />
[ P ]<br />
0<br />
act<br />
0<br />
Solution <strong>of</strong> the three above equations yields a square equation <strong>with</strong> the positive root for<br />
[cx]:<br />
33
[ cx]<br />
( K ([ L]<br />
−[<br />
P ]) + 1)<br />
2<br />
[ L]<br />
+ [ Pact<br />
] 1−<br />
as<br />
act + 4Kas<br />
⋅[<br />
Pact<br />
]<br />
= +<br />
Eq. 3-21<br />
2<br />
2Kas<br />
The total protein concentration [P] is the sum <strong>of</strong> the active fraction [Pact] and the<br />
inactive [Pinact] fraction that does interact <strong>with</strong> the ligand:<br />
[P] = [Pact] + [Pinact]. Eq. 3-22<br />
The initial fluorescence intensity (F(0)) <strong>of</strong> the pure protein is equal:<br />
F(0) = [Pact]⋅φPact_free + [Pinact]⋅φPinact. Eq. 3-23<br />
The observed fluorescence intensity (F) after addition <strong>of</strong> the fluorescent ligand <strong>of</strong> the<br />
fluorescence efficiency φlig-free is equal:<br />
F = [Pact]0⋅φPact_free + [cx]⋅φcx + [L]0⋅φlig_free + [Pinact]⋅φPinact. Eq. 3-24<br />
No assumptions regarding the fluorescence efficiency <strong>of</strong> the inactive protein are necessary.<br />
Simple substitution <strong>of</strong> F(0) and [cx], and definition <strong>of</strong> the difference between the<br />
fluorescence efficiencies <strong>of</strong> the apo-protein and the complex as:<br />
Δφ = φ − − φ<br />
Eq. 3-25<br />
Pact<br />
free<br />
cx<br />
yield the full equation which describes the fluorescence intensity as a function <strong>of</strong> the total<br />
ligand concentration <strong>with</strong>in the course <strong>of</strong> titration:<br />
= − ⋅ Δφ<br />
+ φ + ⋅ φ , Eq. 3-26<br />
F F(<br />
0)<br />
[ cx]<br />
( lig −free)<br />
[ L]<br />
lig −free<br />
This theoretical curve was fitted to the experimental data points. The parameters to be<br />
extracted from the fit were as follows: Kas, [Pact], Δφ, φlig-free, F(0). The latter two<br />
parameters were independently verified experimentally. The accordance between the non-<br />
linearly fitted φlig-free value and the value determined from linear regression to the<br />
fluorescence data for the free ligand at increasing concentrations in the absence <strong>of</strong> the<br />
protein was 4 %. For the experiments at the most elevated temperature, the parameter [Pact]<br />
had a meaning <strong>of</strong> an average value in the course <strong>of</strong> the titration.<br />
eliminated.<br />
Total quenching upon binding was calculated as:<br />
Q = F(<br />
0)<br />
− F(<br />
∞)<br />
= [ Pact<br />
] ⋅ Δφ<br />
. Eq. 3-27<br />
For the peptides, φlig-free was fixed as zero, since the direct tyrosine fluorescence was<br />
The final Kas were calculated as a weighted average <strong>of</strong> 3 to 12 independent titration<br />
series, except for m 7 GTP, for which more than 30 titration experiments were performed, in<br />
order to make statistical analysis <strong>of</strong> the method and as control for each protein batch. The<br />
34
Eadie-H<strong>of</strong>stee representation 187,188 was not used, for its basic assumption <strong>of</strong> a great excess<br />
<strong>of</strong> the ligand in relation to the protein is not satisfied here.<br />
3.5.2. The Gibbs Free Energy <strong>of</strong> Binding<br />
The Gibbs free energy <strong>of</strong> binding <strong>of</strong> cap analogues to <strong>eIF4E</strong> was calculated from Eq.<br />
1-9. The binding constants (Kas) were used for unsymmetrical dinucleotide and<br />
mononucleotide cap analogues, and the microscopic binding constants (Kas (micro) = 0.5 ⋅<br />
Kas) in case <strong>of</strong> the symmetrical cap analogues (m 7 Gpppm 7 G, m 7 Gppppm 7 G, GpppG)<br />
because <strong>of</strong> entropic effects.<br />
3.5.3. Osmotic Stress and Electrostatic <strong>Interaction</strong>s<br />
Increase <strong>of</strong> the KCl concentration at constant pH and temperature is accompanied by<br />
an increasing osmolality (2ϕ[KCl]) and a decreasing activity coefficient <strong>of</strong> water (aw):<br />
2ϕ⋅<br />
[ KCl]<br />
log( a w ) = − , Eq. 3-28<br />
ln10⋅<br />
Ω<br />
where: ϕ = 0 . 91±<br />
0.<br />
01,<br />
an osmotic coefficient <strong>of</strong> KCl approximately constant from 0 to<br />
0.5 M KCl; 189 Ω the number <strong>of</strong> moles <strong>of</strong> free solvent water in 1 kg <strong>of</strong> the solution. The<br />
latter is not assumed to be constant, but the presence <strong>of</strong> other buffer components <strong>of</strong> the<br />
molecular masses μi at the concentrations ci, and the hydration number <strong>of</strong> KCl ( ν ≈ 5),<br />
190<br />
are taken into account:<br />
1000 − μ KCl[<br />
KCl]<br />
− ∑μ<br />
ic<br />
i<br />
Ω =<br />
− ν ⋅[<br />
KCl]<br />
Eq. 3-29<br />
μ<br />
H O<br />
2<br />
Keeping constant Ω = 55.<br />
5 yields overestimation <strong>of</strong> the results concerning the number <strong>of</strong><br />
the water molecules that are exchanged between <strong>eIF4E</strong>-cap complex and the bulk solvent<br />
(ΔN) by ~ 18% (see next page).<br />
3.5.3.1. Davies-Stockes-Robinson Electrostatic Screening Approach<br />
Binding <strong>of</strong> two ions <strong>of</strong> opposite charges (protein z1 and ligand z2) is screened by the<br />
ionic atmosphere <strong>of</strong> the excess salt and other ionized components <strong>of</strong> the solution. The<br />
influence <strong>of</strong> ionic strength and osmotic stress on the activity coefficients <strong>of</strong> the interacting<br />
species leads to the expression for log(Kas) in function <strong>of</strong> [KCl]: 191,192<br />
35
2Az1z<br />
2 I<br />
log( K as ) = log( K as ( 0))<br />
+ + ΔN<br />
log( a w ) , Eq. 3-30<br />
1+<br />
a B I<br />
where ionic strength:<br />
j<br />
2<br />
I = [ KCl]<br />
+ ∑c<br />
iz i , Eq. 3-31<br />
zi is a charge <strong>of</strong> the i-th species, aj is a sum <strong>of</strong> radii <strong>of</strong> interacting species, and ΔN is a<br />
number <strong>of</strong> water molecules taken up (or released, if ΔN < 0) to the macromolecular<br />
surfaces upon complex formation. The temperature-dependent coefficients at 20°C are:<br />
A = 0.<br />
50585,<br />
B = 0.<br />
32789.<br />
192<br />
3.5.3.2. Wyman Linkage <strong>Analysis</strong><br />
Hydration effects as well as ionic interactions are considered stoichiometrically: 193<br />
0<br />
log( K as ) = log( K as ) − c ⋅ log( a KCl)<br />
+ ΔN<br />
⋅ log( a w ) , Eq. 3-32<br />
where c is the total number <strong>of</strong> all ions released from macromolecular surfaces upon<br />
binding. <strong>Thermodynamic</strong> activity <strong>of</strong> KCl (aKCl) is used. Assuming aKCl = [KCl] leads to<br />
underestimation <strong>of</strong> the Kas value extrapolated to 1 M KCl by ~ tw<strong>of</strong>old. Kas 0 corresponds to<br />
the value <strong>of</strong> Kas, when the thermodynamic effects <strong>of</strong> ion release (log([KCl]) = 0 at 1 M<br />
KCl) and <strong>of</strong> the hydration (log(aw) = 0 at 0 M KCl) cancel each other out. Kas 0 occurs at the<br />
KCl concentration <strong>of</strong> approximately 0.3 M. The ion release does not influence the resultant<br />
ionic strength, since the protein and the ligand are present at nano- to micromolar<br />
concentrations, while [KCl] ranges from 0.05 M to 1 M, and the buffer contribution to I at<br />
pH 7.2 is ~ 0.017 M.<br />
The contribution <strong>of</strong> the electrolyte effect (ΔG°el) to the stability <strong>of</strong> the cap-<strong>eIF4E</strong><br />
complex can be calculated from the derivative SKas: 118<br />
as:<br />
SK<br />
as<br />
⎛ ∂ log K as ⎞<br />
≡ ⎜ ⎟<br />
⎝ ∂ log[ KCl]<br />
⎠<br />
T,<br />
p<br />
2φ[<br />
KCl]<br />
= −c<br />
− ΔN<br />
Ω<br />
36<br />
Eq. 3-33<br />
Δ ° = −SK<br />
RT ln[ KCl]<br />
, Eq. 3-34<br />
G el as<br />
and the entropy <strong>of</strong> the electrolyte effect was calculated as:<br />
ΔG°<br />
el<br />
Δ S°<br />
el = − . Eq. 3-35<br />
T
3.5.4. Protonation Equilibria<br />
When the binding event is accompanied by protonation and deprotonation <strong>of</strong> two<br />
specifically interacting residues at constant temperature, ionic strength and osmotic stress,<br />
these processes interfere to produce "bell-like" shaped dependence <strong>of</strong> the binding constant<br />
on pH. In case <strong>of</strong> 7-methylGTP, the population was considered as a mixture <strong>of</strong> cationic<br />
(Lc) and zwitterionic (Lzw) forms:<br />
= [ L ] [ L ] . Eq. 2-36<br />
[ L]<br />
c zw +<br />
The protein was assumed to exist in equilibrium <strong>of</strong> two states, one <strong>with</strong> Glu103 protonated<br />
(Glu) and the other <strong>with</strong> Glu103 deprotonated (Glu), leading to:<br />
−<br />
[ Pact<br />
] = [ Pact<br />
( Glu)]<br />
+ [ Pact<br />
( Glu )] . Eq. 3-37<br />
When the cationic form <strong>of</strong> cap binds to <strong>eIF4E</strong> <strong>with</strong> deprotonated Glu103, the pH-<br />
independent association constant, for the species in their appropriate ionic states, can be<br />
expressed as:<br />
where:<br />
and<br />
−<br />
pH−ind<br />
[ LcPact<br />
( Glu<br />
K as<br />
−<br />
[ Lc<br />
] 0[<br />
Pact<br />
( Glu<br />
[ P<br />
act<br />
c<br />
( Glu<br />
)]<br />
= Eq. 3-38<br />
)]<br />
−<br />
c<br />
c<br />
act<br />
−<br />
0<br />
)] = [ L P ( Glu )] + [ P ( Glu )]<br />
Eq. 3-39<br />
[ L ] = [ L P ( Glu )] + [ L<br />
act<br />
−<br />
c<br />
]<br />
0<br />
act<br />
37<br />
−<br />
0<br />
. Eq. 3-40<br />
The populations <strong>of</strong> both the free protonated ligand ([Lc]0) and the free deprotonated protein<br />
([Pact(Glu − )]0) that actually do not form the complex, are in ionic equilibrium defined by<br />
the effective acidic dissociation constants KL and KP, the former for the ligand in the<br />
presence <strong>of</strong> the protein, and the latter for Glu103 in the presence <strong>of</strong> the cap analogue:<br />
and<br />
K<br />
K<br />
+<br />
[ Lzw<br />
][ H ]<br />
L = Eq. 3-41<br />
[ Lc<br />
] 0<br />
P<br />
[ Pact<br />
( Glu )] 0[<br />
H ]<br />
= Eq. 3-42<br />
[ P ( Glu)]<br />
act<br />
−<br />
The experimentally observed association constant is:<br />
K<br />
+<br />
[ LcPact<br />
( Glu )]<br />
= Eq. 3-43<br />
−<br />
−<br />
([ L]<br />
− [ L P ( Glu )])([ P ( Glu )] −[<br />
L P ( Glu )])<br />
as −<br />
c act<br />
act<br />
c act<br />
−
and after transformation may be expressed as a function <strong>of</strong> pH:<br />
log( K<br />
as<br />
pH−ind<br />
as<br />
pH−pK<br />
L<br />
P<br />
) = log( K ) − log( 1+<br />
10 ) − log( 1+<br />
10 ) , Eq. 3-44<br />
38<br />
pK −pH<br />
where log(Kas pH-ind ), pKL and pKP are fitted parameters. The optimal pH for binding is:<br />
pH opt L P<br />
= ( pK + pK ) / 2 . Eq. 3-45<br />
From the pH-dependence <strong>of</strong> log(Kas), the observed free energy change for the <strong>eIF4E</strong>-<br />
cap association at a given pH can be regarded as:<br />
pH−ind<br />
ΔG<br />
° ( pH)<br />
= ΔG°<br />
+ ΔG°<br />
+ ΔG°<br />
L<br />
P<br />
, Eq. 3-46<br />
where ΔG°L and ΔG°P denote energies <strong>of</strong> bringing <strong>of</strong> the interacting ligand and the protein<br />
residue at a given pH to the appropriate ionic states, for which the binding energy would<br />
equal ΔG° pH-ind :<br />
pH−ind pH−ind<br />
as<br />
Δ G°<br />
= −RT<br />
ln( K ) ; Eq. 3-47<br />
pH−pK<br />
L<br />
Δ G°<br />
= RT ln( 1+<br />
10 ) ; Eq. 3-48<br />
L<br />
pK<br />
pH<br />
Δ G°<br />
= RT ln( 1+<br />
10 ) . Eq. 3-49<br />
P<br />
P −<br />
3.5.5. <strong>Thermodynamic</strong>s<br />
The temperature dependence <strong>of</strong> Kas was analysed according to the van't H<strong>of</strong>f isobaric<br />
equation (Eq. 1-14). 194 The molar heat capacity change ( Δ C ) and the characteristic<br />
temperatures at which<br />
the fitting.<br />
195 196 o<br />
Δ G ,<br />
o<br />
o<br />
Δ S = 0 (TS) or H vH<br />
Eq. 1-9, 1-12, and 1-11, respectvely.<br />
where<br />
o<br />
p<br />
Δ = 0 (TH) were obtained as free parameters <strong>of</strong><br />
o<br />
o<br />
Δ S , and the van't H<strong>of</strong>f enthalpy change H vH<br />
Discrimination between the linear van't H<strong>of</strong>f equation:<br />
ln K<br />
as<br />
o<br />
Δ H vH ,<br />
Snedecor's F-test 197 .<br />
o<br />
o vH<br />
Δ were calculated from<br />
ΔS<br />
ΔH<br />
= − , Eq. 3-50<br />
R RT<br />
o<br />
Δ S = const, and the non-linear model (eq. 4) was based on the statistical<br />
3.5.5.1. Coupling Between Binding and Conformational Transition <strong>of</strong> Ligand<br />
<strong>Thermodynamic</strong> parameters describing intramolecular base stacking <strong>of</strong> dinucleotide<br />
cap-analogues in the cationic form, i.e. entropy ( ΔS°<br />
1 ) and enthalpy ( 1 ΔH°<br />
) changes 198 ,<br />
were used for calculating the following quantities at four temperatures: stacking/unstacking
equilibrium constants ( 1 K ), contributions to enthalpy ( Δ H ), entropy ( Δ S ), and heat<br />
o<br />
psst<br />
capacity ( Δ C ) changes, which result from an induced shift in the self-stacking<br />
equilibrium, and intrinsic enthalpy ( Δ H ) and entropy ( Δ S ) changes <strong>of</strong> the 7-<br />
39<br />
o<br />
0<br />
methylGpppG − <strong>eIF4E</strong> association, according to the equations for the case <strong>of</strong> mandatory<br />
coupling <strong>of</strong> cap-<strong>eIF4E</strong> binding to cap stacking. 122 It is assumed that the entropy changes<br />
are approximately additive.<br />
1<br />
⎛ o<br />
⎜ 1ΔS<br />
1ΔH<br />
K = exp −<br />
⎜<br />
⎝ R RT<br />
o<br />
o<br />
⎞<br />
⎟<br />
⎠<br />
o<br />
sst<br />
o<br />
0<br />
o<br />
sst<br />
Eq. 3-51<br />
o 1ΔH<br />
⋅1<br />
K<br />
ΔHsst = −<br />
Eq. 3-52<br />
1+<br />
K<br />
1<br />
o<br />
o<br />
1ΔH<br />
⋅1<br />
K<br />
ΔSsst = −R<br />
ln(<br />
1+<br />
1K<br />
) −<br />
Eq. 3-53<br />
T<br />
( 1+<br />
K)<br />
1<br />
( ) 2<br />
o 2<br />
o ( 1ΔH<br />
) ⋅1<br />
K<br />
ΔCp = −<br />
Eq. 3-54<br />
sst 2<br />
RT 1+<br />
1K<br />
o<br />
0<br />
o<br />
cal ion<br />
o<br />
sst<br />
ΔH = ΔH<br />
− − ΔH<br />
Eq. 3-55<br />
o o o<br />
ΔS0 = ΔS<br />
− ΔSsst<br />
Eq. 3-56<br />
3.5.5.2. Enthalpy-Entropy Compensation in Congener Series – General Model 199<br />
For a system in which the number <strong>of</strong> states <strong>with</strong> an energy from U to U+δU is<br />
ω(U)δU, the mean energy (which is approximately the enthalpy <strong>of</strong> the system) is:<br />
∫ U ⋅ ϖ(<br />
U)<br />
⋅ e<br />
E = U =<br />
∞<br />
∫ ϖ(<br />
U)<br />
⋅ e<br />
−∞<br />
−U<br />
kT<br />
−U<br />
kT<br />
dU<br />
dU<br />
Eq. 3-57<br />
If some energy levels <strong>with</strong>in energy range from U' to U'+δU are significantly<br />
perturbed by ΔU > 3 kT, then the change in the energy due to the perturbance is:<br />
where<br />
ΔE ≈ (E − U')⋅P(U')dU, Eq. 3-58<br />
P(<br />
U'<br />
)<br />
−U'<br />
kT<br />
= ϖ(<br />
U'<br />
) ⋅ e is the probability <strong>of</strong> finding the system in a state <strong>with</strong> energy U'<br />
in the unperturbed system. Large and complex protein systems have many closely spaced<br />
energy levels, i. e. P(U')dU
ΔG ≈ kT⋅P(U')dU. Eq. 3-59<br />
The entropy change is then:<br />
TΔS ≈ (E − U' − kT)⋅P(U')dU. Eq. 3-60<br />
Enthalpy-entropy compensation can be thus described by a compensation temperature (Tc)<br />
that is related to the difference between the mean energy and the energy <strong>of</strong> the perturbed<br />
states:<br />
T c<br />
ΔH°<br />
ΔE°<br />
T<br />
= ≈ ≈<br />
Eq. 3-61<br />
ΔS°<br />
ΔS°<br />
RT<br />
1 −<br />
E°<br />
− U°<br />
'<br />
3.6. Isothermal Titration Calorimetry<br />
Microcalorimetry provides a direct route to thermodynamic characterization <strong>of</strong><br />
bimolecular equilibrium interactions. 200 When heat is generated or absorbed <strong>with</strong>in the<br />
sample cell a compensation appears in the feedback power to equilibrate the temperatures<br />
in the sample cell and in the reference cell. The time integral <strong>of</strong> the power deflection is a<br />
measure <strong>of</strong> the heat. The molar calorimetric enthalpy (ΔH°cal) and the association constant<br />
could be determined in one experiment by direct measurement <strong>of</strong> the heat <strong>of</strong> interaction<br />
when one component is titrated into the other. However, two conditions must be<br />
simultaneously satisfied so that determination <strong>of</strong> Kas is reliable and accurate: 201<br />
1. A product <strong>of</strong> the number <strong>of</strong> the interacting molecules ([Pact] expressed in M) and the<br />
association constant (Kas expressed in M -1 ) should be in the order <strong>of</strong> 100.<br />
2. Each injection should have at least 40 μJ <strong>of</strong> heat exchanged <strong>with</strong> the cell <strong>of</strong> the<br />
microcalorimeter.<br />
It was entirely impossible to meet these conditions for <strong>eIF4E</strong> and those cap analogues,<br />
which were described by the non-linear van't H<strong>of</strong>f plots. Numerical simulations <strong>of</strong> such<br />
experiments for m3 2,2,7 GTP <strong>with</strong> added Gaussian noise at the lowest possible level showed<br />
that fitting <strong>of</strong> the binding curve would be hopeless. Thus, a modified "single injection"<br />
method was proposed to obtain the most accurate values <strong>of</strong> the molar calorimetric<br />
enthalpies at several temperatures.<br />
40
3.6.1. Calorimetric Measurements for m 7 GpppG<br />
ITC experiments were run on OMEGA Ultrasensitive Titration Calorimeter<br />
(MicroCal, MA, U.S.A.), calibrated by 18-crown-6 titration <strong>with</strong> BaCl2. * The jacket <strong>of</strong> the<br />
microcalorimeter was filled <strong>with</strong> dry nitrogen to prevent condensation on the outer surface<br />
<strong>of</strong> the cells below room temperature. A refrigerated circulated bath (7 °C) was connected<br />
to the microcalorimeter to keep the surroundings <strong>of</strong> the cells cooler than the temperature <strong>of</strong><br />
a given experiment. The reference cell was filled <strong>with</strong> deionized water. Slow stirring at 240<br />
rpm was applied to avoid protein precipitation. The system was allowed to equilibrate until<br />
a stable baseline was observed (≥ 30 min), before an automated titration was initiated.<br />
Suitable buffers (50 mM Hepes/KOH, 100 mM KCl, 1 mM EDTA) were prepared to<br />
keep pH 7.20 ± 0.02 at 288.1, 293.1, 298.4, and 303.2 K. The protein sample buffer was<br />
exchanged by 4-fold centrifugation on 5 kDa Centricon filters (Millipore, MA, USA).<br />
After last centrifugation, the flow-through buffer was collected to dissolve 7-methylGpppG<br />
and to make control measurements <strong>of</strong> heat <strong>of</strong> the ligand dilution in the buffer. The<br />
concentration <strong>of</strong> the injected ligand was 1.00 ± 0.07 mM in each case. Samples were<br />
degassed, then filtered through 0.22 μm filter (Millipore, USA) directly before using. The<br />
main part from each protein solution was used for ITC and the remaining part for the<br />
control fluorescence titration. This ensured consistency for all the measurements. The<br />
active protein concentrations at four temperatures were 8.97 μM, 7.42 μM, 3.61 μM and<br />
5.35 μM, respectively.<br />
3.6.1.1. Modified "Single Injection" Experiment<br />
Low solubility <strong>of</strong> <strong>eIF4E</strong> (28-217) hampered also direct determination <strong>of</strong> Kas for 7-<br />
methylGpppG by the ITC measurements. The very first injections into the <strong>eIF4E</strong> solution<br />
results in ~ 35 μJ <strong>of</strong> the evolved heat, so at the verge <strong>of</strong> the instrument sensitivity, ~ 40<br />
μJ. 201 The subsequent heat signals decrease <strong>with</strong> the course <strong>of</strong> the titration due to the<br />
negative value <strong>of</strong> ΔH°, thus becoming indiscernible from the noise. The titrations were<br />
performed several times for different injection volumes (data not shown) but<br />
unsuccessfully. However, it was possible to determine the calorimetric enthalpies by a<br />
modified "single injection" method. The ligand solution was injected into the calorimetric<br />
cell (1386 μl volume) filled <strong>with</strong> <strong>eIF4E</strong> solution. Next, the 7-methylGpppG solution was<br />
* Calibration was done by Dr. Ma³gorzata Wszelaka-Rylik (Inst. Phys. Chem. PAS, Warszawa)<br />
41
injected into the buffer to measure the heat <strong>of</strong> dilution. Each experiment consisted <strong>of</strong> the<br />
main 40 μl injection, preceded by two 1 μl injections to calculate the correction for the<br />
initial outflow from the syringe, and followed by two 4 μl injections to check the protein<br />
saturation <strong>with</strong> the ligand. It was therefore possible to determine the total emitted heat in<br />
the most reliable way.<br />
3.6.2. Calorimetric Data Treatment<br />
After integration <strong>of</strong> all signals, the corresponding values from the protein titration<br />
<strong>with</strong> the ligand, and from the buffer titration <strong>with</strong> the ligand were subtracted from each<br />
other to yield the total calorimetric enthalpy<br />
o<br />
Δ H . As the heat which is exchanged at the<br />
42<br />
μl injections,<br />
which was apparently decreased by the common instrumental artifact (leakage from the<br />
syringe) during the baseline equilibration.<br />
Calorimetric<br />
<strong>of</strong> the enthalpies:<br />
o<br />
p<br />
o cal<br />
3.6.2.1. Buffer Ionization Heats<br />
o<br />
ion<br />
o o<br />
o o<br />
Δ Hion<br />
, Hcal ion = ΔHcal<br />
− ΔHion<br />
Δ − (see below).<br />
o Buffer ionization heats ( Δ Hion<br />
) <strong>of</strong> Hepes at pH =7.2 were calculated as:<br />
−pKa<br />
10<br />
−pKa<br />
−pH<br />
o<br />
H−diss<br />
ΔH =<br />
⋅ ΔH<br />
Eq. 3-62<br />
10 + 10<br />
o from the molar ionization heat for Hepes, H H−diss<br />
o temperature-dependence <strong>of</strong> H H−diss<br />
Δ = +20.95 kJ⋅mol -1 at 298.2 K and<br />
o<br />
Δ estimated as δ Δ H H−diss<br />
/δT = +0.0648 kJ⋅mol -1 K -1 , 202<br />
and pKa = 7.35 ± 0.05 for 7-methylGpppG. 177 The pKa changes were negligible over the<br />
temperature range used.<br />
3.7. NMR Spectroscopy<br />
Nuclear magnetic resonance (NMR) spectroscopy 203 is concerned <strong>with</strong> the magnetic<br />
energy <strong>of</strong> nuclei in a strong, static, magnetic field <strong>of</strong> strength Bo, (2 T to 21 T), and the<br />
transitions in the wave region <strong>of</strong> 2 - 900 MHz. Significance <strong>of</strong> the NMR spectroscopy in<br />
dynamic studies <strong>of</strong> molecules is related to interaction <strong>of</strong> a nucleus <strong>with</strong> surrounding
electrons (chemical shift) and other nuclei via the electrons (scalar coupling). The former<br />
leads to shifts <strong>of</strong> the resonance frequencies <strong>of</strong> the nuclei located in different chemical<br />
environment since the magnetic field is shielded by the electrons to B = Bo(1-σ), where σ<br />
is the shielding constant. Scalar coupling results in splitting <strong>of</strong> the resonance lines<br />
according to values <strong>of</strong> the coupling constants for the nuclear pairs.<br />
Formation <strong>of</strong> molecular complexes can be followed by changes <strong>of</strong> the chemical shifts<br />
<strong>of</strong> the nuclei directly engaged in the interactions. The total chemical shift can be<br />
represented as a sum <strong>of</strong> local effects (diamagnetic, d, and paramagnetic, p, contributions)<br />
and long-range effects, direct anisotropy (a) <strong>of</strong> the surrounding chemical bonds, ring<br />
currents (r), electric field (e) and solvent (s):<br />
δ = δd(local) + δp(local) + δa + δr + δe + δs<br />
43<br />
Eq. 3-63<br />
Substantial deshielding effect observed for protons upon formation a hydrogen bond<br />
(high value <strong>of</strong> δ) arises from an interplay <strong>of</strong> the increased electron density in the vicinity <strong>of</strong><br />
the proton (increase <strong>of</strong> δd(local)) and predominating effects <strong>of</strong> the additional restraint<br />
placed on field-induced electron circulations by the acceptor (increase <strong>of</strong> δp(local)) and its<br />
electric field effect (δe). Stacking <strong>of</strong> π-electron rings is usually accompanied by an upfield<br />
shift <strong>of</strong> the protons attached to the rings (δr), since the additional magnetic field from the<br />
circulating π-electrons reinforces Bo in the planes <strong>of</strong> the rings.<br />
To detect the NMR signal <strong>of</strong> the exchangeable protons in aqueous solution, e.g. H(8)<br />
<strong>of</strong> m 7 G, H2O instead <strong>of</strong> 2 H2O must be applied. The strong water signal in the 1 H NMR<br />
measurements needs to be suppressed.<br />
3.7.1. NMR Spectra Recording<br />
1 H NMR spectra <strong>of</strong> tryptophan N-acetylamid, a dodecapeptide DGIEPMWEDEKN,<br />
and cap analogues were recorded on a VarianUNITYplus 500 MHz spectrometer, in 1/15<br />
M phosphate buffer, pH 5.6 or 5.2, <strong>with</strong> sodium 3-trimethylsilyl-[2,2,3,3- 2 H4]propionate<br />
(TSP) as internal standard, and 10% 2 H2O for spin locking. The assignment <strong>of</strong> protons in<br />
the cap analogues and tryptophan were made on the basis <strong>of</strong> the splitting patterns <strong>of</strong> the<br />
proton resonances and their chemical shifts values (accuracy <strong>of</strong> ± 0.001 ppm). The<br />
unambiguous assignment for the H(4)/H(7) and H(5)/H(6) proton pairs in the tryptophan<br />
indole ring were done earlier from analysis <strong>of</strong> the cross-peaks in 2D ROESY spectrum by<br />
Dr. Ryszard Stolarski. 204 An effective method (~1000-fold suppression) <strong>of</strong> a binominal
1331 experiment was used for water suppression. 205 It tailors the excitation pr<strong>of</strong>iles so that<br />
there is zero net excitation <strong>of</strong> water. The hard pulse sequence was p1-τ-p2-τ-p3-τ-p4 <strong>with</strong><br />
the length ratio 1 : 3 : 3 : 1. The sum <strong>of</strong> the pulse lengths was 50 μs giving rotation <strong>of</strong> the<br />
proton magnetization by π/2 at B1 <strong>of</strong> 35 dB (attenuation <strong>of</strong> the maximal B1 strength). The<br />
<strong>of</strong>fset <strong>of</strong> the maximal excitation in respect to the carrier frequency put on the water signal,<br />
determined by the period τ, was 1500 Hz for maximal excitation in the aromatic region.<br />
3.7.2. NMR Data <strong>Analysis</strong><br />
3.7.2.1. Concentration Dependence<br />
Observed differences <strong>of</strong> 1 H chemical shifts (Δδ = δ(mixture) - δ(free)) <strong>of</strong> tryptophan<br />
N-acetylamid or <strong>of</strong> the dodecapeptide tryptophan (concentration ctrp) due to stacking upon<br />
titration <strong>with</strong> a cap analogue at 298 K depend on the total concentration <strong>of</strong> the cap<br />
analogue (ccap) according to equation:<br />
Δδ<br />
= Δδ<br />
u<br />
c<br />
⋅<br />
c<br />
u<br />
trp<br />
+ Δδ<br />
st<br />
⎛<br />
⎜<br />
c<br />
⋅ 1−<br />
⎜<br />
⎝<br />
c<br />
u<br />
trp<br />
⎞<br />
⎟ , Eq. 3-64<br />
⎟<br />
⎠<br />
where Δδu is the difference <strong>of</strong> the chemical shift <strong>of</strong> the unstacked tryptophan proton (fixed<br />
as 0), Δδst is the difference <strong>of</strong> the chemical shift <strong>of</strong> the stacked tryptophan proton (fitted as<br />
a free parameter), and equilibrium concentration <strong>of</strong> the unbound tryptophan proton (cu) is<br />
determined by a fitted association constant (K):<br />
c<br />
u<br />
2<br />
2<br />
K ⋅(<br />
ctrp<br />
− ccap<br />
) −1+<br />
K ⋅(<br />
ctrp<br />
− ccap<br />
) + 2⋅<br />
K(<br />
ctrp<br />
+ ccap)<br />
+ 1<br />
= Eq. 3-65<br />
2⋅<br />
K<br />
3.7.2.2. Temperature Dependence<br />
Temperature dependence <strong>of</strong> observed differences <strong>of</strong> 1 H chemical shifts <strong>of</strong> tryptophan<br />
N-acetylamid at 1 mM in the presence <strong>of</strong> 29.8 mM 7-methylGMP, and <strong>of</strong> the<br />
dodecapeptide tryptophan at 2.8 mM in the presence <strong>of</strong> 17.2 mM 7-methylGpppG is<br />
described by equation:<br />
Δδ<br />
= Δδ<br />
st<br />
⎛ ⎞<br />
⎜<br />
cu<br />
⋅ 1 − ⎟ , Eq. 3-66<br />
⎜ ⎟<br />
⎝<br />
ctrp<br />
⎠<br />
where cu is defined above, but the association constant is temperature dependent according<br />
to the van't H<strong>of</strong>f isobaric equation:<br />
44
⎛ ΔS°<br />
ΔH°<br />
vH ⎞<br />
K = exp⎜<br />
− ⎟ , Eq. 3-67<br />
⎝ R R ⋅T<br />
⎠<br />
R – the gas constant, T – absolute temperature. The standard molar entropy change (ΔS°)<br />
and enthalpy change (ΔH°vH) are fitted, constant parameters or treated as functions <strong>of</strong><br />
temperature. Then the fitted parameter is the heat capacity change ( Δ C ) that is involved<br />
through Eq. 1-11 and 1-12.<br />
3.8. Dynamic Light Scattering<br />
Light is scattered by interactions <strong>of</strong> electrons <strong>of</strong> molecules dissolved in a solution<br />
<strong>with</strong> the incident radiation. The scattering is quasi-elastic. The oscillating electric field<br />
causes a vibration <strong>of</strong> electrons turning them into oscillating dipoles which reemit radiation.<br />
The power spectrum is broadened in the frequency domain due to the Doppler effect on the<br />
Brownian motion <strong>of</strong> scattering macromolecules and hence due to their diffusion coefficient<br />
(D). D is related to the size <strong>of</strong> the molecules, for instance to the hydrodynamic radius (Rh)<br />
for approximately globular proteins. Fluctuations in the intensity <strong>of</strong> light scattered by a<br />
small volume <strong>of</strong> a solution in the microsecond time-scale are described by an<br />
autocorrelation function (g1(t)) which is registered by the instrument. For small<br />
monodispersed particles and homogeneous spheres the normalized autocorrelation function<br />
<strong>of</strong> scattered electric field is: 206<br />
g<br />
1<br />
( t)<br />
v<br />
−DQ<br />
2<br />
t<br />
= e , Eq. 3-68<br />
where Q v is the scattering vector resulting from the difference <strong>of</strong> the scattered and the<br />
incident wave vectors, t is the time interval for displacement <strong>of</strong> the scattering particle. D is<br />
related to the reciprocal <strong>of</strong> the characteristic decay time (Γ):<br />
2<br />
DQ v<br />
Γ =<br />
Eq. 3-69<br />
For a continuous polydispersed systems <strong>with</strong> the Γ distribution function (G(Γ)):<br />
∞<br />
−Γt g1<br />
( t)<br />
= a ∫ G(<br />
Γ)<br />
e dΓ<br />
, where a = const. Eq. 3-70<br />
0<br />
An inverse Laplace transform generates the distribution <strong>of</strong> decay rates (G(Γ)), from which<br />
the diffusion coefficients distribution and next the particle sizes distribution can be<br />
determined using the Stokes-Einstein equation:<br />
45<br />
o<br />
p
k BT<br />
D = Eq. 3-71<br />
6πηR<br />
h<br />
Molecular weight calculations are performed by means <strong>of</strong> a logarithmic function<br />
based upon experimental data on several well-characterized proteins, provided by the<br />
manufacturer <strong>of</strong> the DLS equipment.<br />
The DLS measurements were run on a DynaPro-801 Molecular Size Detector<br />
(<strong>Protein</strong> Solutions Inc., Charlottesville, VA) for <strong>eIF4E</strong> (residues 33-217) at a concentration<br />
<strong>of</strong> 1 mg/ml, in the absence and in the presence <strong>of</strong> 50-fold excess <strong>of</strong> m 7 GTP, p-Cl-bz 7 GTP,<br />
m3 227 GTP, m 7 GpppG, m 7 GppppG, or m 7 Gppppm 7 G, at 20ºC, in the standard buffer.<br />
3.9. Statistical <strong>Analysis</strong><br />
Goodness <strong>of</strong> fits was described by the R 2 value, which was not less than 0.999 and 0.99 for<br />
binding isotherms <strong>of</strong> cap analogues and peptides, respectively:<br />
∑ ( y exp − yfit<br />
)<br />
2<br />
R = 1−<br />
, Eq. 3-72<br />
2<br />
∑(<br />
y − y)<br />
exp<br />
2<br />
where yexp are measured values, yfit are fitted values, and y is the mean <strong>of</strong> the measured<br />
values. Other R 2 values are given in the text.<br />
Statistical analysis was done on the basis <strong>of</strong> the runs test and the P value 197 which<br />
denotes the probability that distribution <strong>of</strong> the fitting residuals <strong>of</strong> the y-values is random<br />
and does not depend on the x-values.<br />
Discrimination between two models <strong>of</strong> different numbers <strong>of</strong> degrees <strong>of</strong> freedom (ν1<br />
and ν2) was based <strong>of</strong> the statistical two-parameter Snedecor's F-test. 197 The test allows for<br />
a consistent and systematic assessment whether the results obtained from more<br />
complicated model <strong>with</strong> a greater number <strong>of</strong> fitted parameters are statistically important in<br />
comparison <strong>with</strong> the simpler model. The F-ratio quantifies the relationship between the<br />
relative decrease in sum-<strong>of</strong>-squares and the relative decrease in degrees <strong>of</strong> freedom:<br />
F =<br />
2<br />
2<br />
( ∑( y − y ) − ∑(<br />
y − y ) )<br />
exp<br />
fit _1<br />
exp<br />
fit _ 2<br />
( ν1<br />
− ν2<br />
) ν2<br />
46<br />
∑(<br />
y<br />
exp<br />
− y<br />
fit _ 2<br />
)<br />
2<br />
Eq. 3-73<br />
The P(ν1, ν2) value in this case is the probability that the improvement <strong>of</strong> the fit <strong>with</strong> the<br />
greater number <strong>of</strong> fitted parameters (i. e. <strong>with</strong> smaller ν) expressed by the F-ratio could be<br />
obtained randomly. The significance level was assumed as P(ν1, ν2) < 0.1 or < 0.05.
Errors <strong>of</strong> reported values (one standard deviation) were calculated according to the<br />
appropriate propagation rules 207 on the basis <strong>of</strong> the numerical uncertainty resulting from<br />
the fitting. Thanks to avoidance <strong>of</strong> linear transformations <strong>of</strong> the fluorescence titration data,<br />
their experimental uncertainties were constant <strong>with</strong>in the whole range <strong>of</strong> x-axis and almost<br />
always negligibly small (less than the size <strong>of</strong> graphical symbols). Hence, they did not<br />
influence much the final results (Kas). Experimental errors were taken into account on<br />
integration <strong>of</strong> the calorimetric data and on analyses <strong>of</strong> thermodynamic parameters.<br />
Regressions were performed by means <strong>of</strong> linear or non-linear, least-squares method,<br />
using PRISM 3.02 from GraphPad S<strong>of</strong>tware Inc., U.S.A. or ORIGIN 6.0 from Microcal<br />
S<strong>of</strong>tware Inc., U.S.A.<br />
3.10. Additional Information − Crystallography<br />
The crystallographic structure <strong>of</strong> murine <strong>eIF4E</strong> (residues 28-217) in complex <strong>with</strong> 7-<br />
methyl-GpppG to which the thesis <strong>of</strong>ten refers was resolved by Drs. Joseph Marcotrigiano<br />
and Stephen K. Burley (The Rockefeller University, N.Y., U.S.A.) similarly as reported<br />
previously 75 and was published in a joint paper. 1 Atomic coordinates have been submitted<br />
to the protein databank, accession code: 1L8B.<br />
47
4. Results and Discussion<br />
4.1. Methodological Aspects <strong>of</strong> Studies <strong>of</strong> Non-Enzymatic<br />
<strong>Protein</strong><br />
Biophysical analysis <strong>of</strong> intermolecular interactions aimed at rational design <strong>of</strong><br />
therapeutic agents requires reliable measurements <strong>of</strong> the protein-ligand association in<br />
solution as a necessary counterpart to high resolution structural studies. For <strong>eIF4E</strong>, such<br />
consistent information was lacking. The association constants have been changing<br />
significantly <strong>with</strong> each new publication due to several neglected sources <strong>of</strong> errors, both<br />
experimental and numerical. First, the lack <strong>of</strong> quantitative control <strong>of</strong> the protein activity.<br />
All association constants (Kas) reported in the previous studies were derived assuming<br />
100% activity <strong>of</strong> the protein. 5,156,161-164 As <strong>eIF4E</strong> is known to be a highly unstable protein,<br />
the prevailing assumption that entire population <strong>of</strong> <strong>eIF4E</strong> is active at every conditions was<br />
groundless. Second, the presence <strong>of</strong> practically unremovable contamination <strong>of</strong> affinity-<br />
purified <strong>eIF4E</strong> <strong>with</strong> the cap-analogue that was used for the elution from the cap-affinity<br />
column. Such <strong>eIF4E</strong> can be up to 60% m 7 GTP-bound, even after application <strong>of</strong> ionic<br />
exchange chromatography 208 or repeated buffer exchange on a 5 kDa NMWL filter. Third,<br />
dilution <strong>of</strong> a sample in the course <strong>of</strong> the titration, inner filter effect, and contribution <strong>of</strong> a<br />
free ligand to total fluorescence, were only partially eliminated in the previous<br />
publications. Dilution was considered in most <strong>of</strong> them, 5,156,161-163 while the inner filter<br />
effect and estimated emission <strong>of</strong> a free ligand were taken into account properly in the<br />
single paper. 5 The results <strong>of</strong> studies on <strong>eIF4E</strong> fused <strong>with</strong> GST, which molecular mass is<br />
comparable <strong>with</strong> that <strong>of</strong> <strong>eIF4E</strong>, were useful only for qualitative interpretation. 164<br />
All above-mentioned experimental problems are rigorously analysed herein.<br />
Purification <strong>of</strong> <strong>eIF4E</strong> via refolding from inclusion bodies guarantees that the protein is cap-<br />
free. Since <strong>eIF4E</strong> is a non-enzymatic protein, it is impossible to determine the specific<br />
activity <strong>of</strong> each sample from independent experiments. The active protein concentration<br />
([Pact]) is thus introduced as a free parameter in the numerical analysis. Addition <strong>of</strong> the<br />
next fitting parameter requires a novel, very precise measuring method <strong>of</strong> fluorescence<br />
quenching. <strong>Interaction</strong>s <strong>of</strong> the apo-protein <strong>with</strong> cap analogues are studied by the time-<br />
synchronized titration, yielding the goodness <strong>of</strong> fit R 2 = 0.999-0.9999, what assures the<br />
48
accuracy enough to extract the lacking information on the actual protein activity from each<br />
specific titration. Only this approach, combining a rigorous gathering <strong>of</strong> experimental data<br />
<strong>with</strong> simultaneous check-up on the protein activity, provides results <strong>with</strong>out systematic<br />
deviation from the theoretical model (P 0.05), and leads to concordance among Kas and<br />
other fitted parameters.<br />
4.1.1. Solution <strong>of</strong> Spectroscopic Difficulties<br />
Absorption<br />
0.012<br />
0.009<br />
0.006<br />
0.003<br />
0.000<br />
250 280 310 340 370<br />
λ [nm]<br />
Figure 4-1. Absorption (left) and fluorescence (right, λex = 280 nm) spectra <strong>of</strong> <strong>eIF4E</strong> (solid line,<br />
0.2μM) and m 7 GTP (dotted line, 4μM) at 20°C. Typical <strong>eIF4E</strong> fluorescence quenching spectra<br />
upon increasing concentration <strong>of</strong> m 7 GTP (0.7nM−4μM). Fluorescence spectra for higher<br />
concentrations <strong>of</strong> m 7 GTP exhibit an increasing emission <strong>of</strong> free m 7 GTP present in solution.<br />
All fluorescent species in solution are explicitly included in the numerical analysis.<br />
The complete overlapping <strong>of</strong> the absorption spectra for <strong>eIF4E</strong> and the cap analogues (Fig.<br />
4-1) makes it impossible to excite the protein selectively. The emission spectra are also<br />
overlapped; therefore the contribution from the increasing concentration <strong>of</strong> the free cap<br />
analogues to the total fluorescence in the course <strong>of</strong> the titration must not be neglected. This<br />
makes the spectroscopic analysis <strong>of</strong> intermolecular interactions complicated. Subtraction <strong>of</strong><br />
the free cap fluorescence is groundless, since the equilibrium concentration <strong>of</strong> the free<br />
ligand depends on the association constant to be determined. The spectra overlapping leads<br />
to erroneous attribution <strong>of</strong> the signal plateau to the saturation state, while the apparent<br />
plateau originates from two effects that cancel each other out: the quenching <strong>of</strong> the<br />
49<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Fluorescence a. u.
intrinsic protein fluorescence and the increasing emission <strong>of</strong> the free cap analogues. Both<br />
effects are now taken into account. As a result, the observed maximal fluorescence<br />
quenching is different for various cap analogues (Fig. 4-2), and smaller than the calculated<br />
intrinsic <strong>eIF4E</strong> fluorescence quenching (Q, see Eq. 3-27, p. 34), which is the same for<br />
different cap analogues (~ 65% for the fresh protein). The association constants determined<br />
from the titration curves <strong>with</strong> the neglected free ligand emission would be erroneous,<br />
especially for weakly binding and strongly emitting ligands. The present method <strong>of</strong> data<br />
analysis eliminates these problems.<br />
Fluorescence (a. u.)<br />
Residuals<br />
100<br />
80<br />
60<br />
40<br />
20<br />
1<br />
-1<br />
10 -3 10 -2 10 -1 10 0 10 1 10 2<br />
cap concentration (μM) (μ<br />
Figure 4-2. Titration curves for m 7 GTP ( ), m 7 GpppG ( ), and m3 2,2,7 GTP ( ) at 20°C, and<br />
fitting residuals. An increasing fluorescence signal at higher cap concentrations originates from<br />
emission <strong>of</strong> the free-cap.<br />
50
4.1.2. Activity <strong>of</strong> Non-Enzymatic <strong>Protein</strong><br />
Quantitative control <strong>of</strong> the protein activity belongs to the fundamental state-<strong>of</strong>-the-art<br />
biochemical methods usually applied to enzymatic proteins, and was to date impossible to<br />
do <strong>with</strong> non-enzymatic proteins. Determination <strong>of</strong> the actual concentration <strong>of</strong> the active<br />
<strong>eIF4E</strong> factor upon each titration was the key, which made it possible to interpret the results<br />
<strong>of</strong> the binding studies in a quantitative manner.<br />
For previously frozen samples <strong>of</strong> apo-protein, the active fraction decreases down to<br />
even less than 10 % (Table 4-1, Fig. 4-3). The inactive fraction aggregates and precipitates,<br />
in spite <strong>of</strong> low protein concentration <strong>of</strong> the stored stock solution (< 0.5 mg/ml), the<br />
presence <strong>of</strong> 10 % (v/v) glycerol, 0.5 mM EDTA, and flash freezing <strong>of</strong> the aliquots (from<br />
10 μL to 50 μL). This is in contrast to the cap-saturated <strong>eIF4E</strong> that is stabilized by the<br />
ligand (see 4.2.3.1.6.1., p. 82). The experiments reported in this thesis were performed<br />
temporarily <strong>with</strong> frozen protein and then repeated <strong>with</strong> freshly prepared <strong>eIF4E</strong> (not frozen,<br />
stored at 4 °C), for which the quantity <strong>of</strong> active protein was satisfactory and varied mostly<br />
from 80 % to 113 % (± 12%) <strong>of</strong> the concentration estimated from the absorption spectra<br />
(Table 4-1). The statistics <strong>of</strong> the results proves that determination <strong>of</strong> the Kas values is<br />
reliable, and is unaffected by both the actual concentration <strong>of</strong> the active protein in the<br />
sample and the accompanying amount <strong>of</strong> the inactive protein. This indicates that the<br />
inactivation by freezing, e. g. by cold denaturation, 137 or by elevated temperatures during<br />
titrations do not partially diminish the affinity <strong>of</strong> the whole protein population, but removes<br />
a part <strong>of</strong> the population from the binding reaction, while the native protein molecules still<br />
retain their specific affinity.<br />
51
Table 4-1. Concentration <strong>of</strong> the active <strong>eIF4E</strong> protein ([Pact]) in the cuvette during fluorescence<br />
titration <strong>with</strong> m 7 GTP, equilibrium association constants (Kas), and percentage <strong>of</strong> the active protein<br />
in respect to the total concentration estimated from absorbance <strong>of</strong> the fresh or frozen stock solution<br />
([Ptotal]), at 20 °C, pH 7.2, 100 mM KCl.<br />
[Pact] (μM) Kas ⋅ 10 -6 (M -1 ) [Pact]/[Ptotal] (%)<br />
Fresh protein<br />
0.0283 ± 0.0023 115 ± 14 28<br />
0.0405 ± 0.0091 179 ± 103 110<br />
0.1026 ± 0.0053 106 ± 19 59<br />
0.1426 ± 0.0028 143 ± 18 93<br />
0.1551 ± 0.0037 72.3 ± 6.9 80<br />
0.1720 ± 0.0057 113 ± 24 105<br />
0.1798 ± 0.0014 103.2 ± 5.1 90<br />
0.1861 ± 0.0074 76 ± 15 113<br />
0.1893 ± 0.0056 108 ± 23 89<br />
0.2049 ± 0.0013 113.3 ± 6.0 103<br />
0.2258 ± 0.0067 158 ± 47 98<br />
0.2990 ± 0.0075 95 ± 23 60<br />
Frozen protein<br />
0.0005 ± 0.0053 89 ± 26 0.2<br />
0.0167 ± 0.0013 190 ± 19 4.0<br />
0.0239 ± 0.0012 83.3 ± 4.6 8.1<br />
0.0254 ± 0.0025 129 ± 18 20<br />
0.0265 ± 0.0037 70 ± 10 6.8<br />
0.0385 ± 0.0020 157 ± 23 21<br />
0.0466 ± 0.0035 111 ± 19 18<br />
0.0677 ± 0.0054 93 ± 24 53<br />
0.0720 ± 0.0022 75.9 ± 6.9 19<br />
0.0762 ± 0.0034 170 ± 44 42<br />
0.0867 ± 0.0029 118 ± 15 43<br />
0.0967 ± 0.0140 125 ± 71 76<br />
0.1896 ± 0.0180 73 ± 34 94<br />
52
Active protein (%)<br />
N<br />
100<br />
50<br />
10<br />
0<br />
0.0 0.1 0.2 0.3 0.4 0.5<br />
8<br />
6<br />
4<br />
2<br />
0<br />
Total protein concentration (μM)<br />
70 90 110 130 150 170 190<br />
K as ⋅ 10 -6 (M -1 )<br />
(a) (b)<br />
(c) (d)<br />
Figure 4-3. Statistics <strong>of</strong> titration results obtained for frozen ( ), and fresh protein () from one<br />
purification batch, used <strong>with</strong>in several subsequent days, at 20°C. (a) Active protein fraction<br />
([Pact]/[Ptotal]) vs total protein concentration during fluorescence titration. The frozen protein tends<br />
to precipitate the more the higher is the concentration <strong>of</strong> the solution, while [Pact]/[Ptotal] <strong>of</strong> the fresh<br />
samples seems to be clustered about 100% activity. (b) Values <strong>of</strong> association constant (Kas) for<br />
m 7 GTP binding to the active fraction <strong>of</strong> <strong>eIF4E</strong> are invariant irrespectively <strong>of</strong> the presence <strong>of</strong><br />
various amount <strong>of</strong> inactive protein in the frozen or fresh samples. (c) Distribution (N) <strong>of</strong> Kas values<br />
obtained from 26 repeats <strong>of</strong> titration experiments. (d) Gaussian distributions <strong>of</strong> the Gibbs free<br />
energy <strong>of</strong> binding <strong>of</strong> m 7 GTP to frozen ( , thin solid line) and fresh ( , broken line) <strong>eIF4E</strong>;<br />
summary ( , thick solid line, = -45.11 kJ/mol, SD = 0.87 kJ/mol).<br />
The influence <strong>of</strong> the concentration <strong>of</strong> active protein as fitting parameter on quality <strong>of</strong><br />
final results is illustrated in Fig. 4-4. Titration <strong>of</strong> a frozen sample <strong>with</strong> m 7 GTP in the<br />
nanomolar concentration range, i.e. where fluorescence <strong>of</strong> the ligand is negligible at the<br />
spectral bandwidths suitable for observation <strong>of</strong> the protein fluorescence changes, allows for<br />
analysis <strong>of</strong> the protein activity in the simple case <strong>with</strong> φlig-free = 0.<br />
53<br />
Kas (M -1 )<br />
N<br />
10 9<br />
10 8<br />
10 7<br />
16<br />
12<br />
8<br />
4<br />
0 20 40 60 80 100 120<br />
Active protein (%)<br />
0<br />
-47 -46 -45 -44 -43<br />
ΔG° (kJ/mol)
Fluorescence (a. u.)<br />
Residuals<br />
800<br />
600<br />
400<br />
200<br />
8<br />
0<br />
-8<br />
(a) (b)<br />
10 -3 10 -2 10 -1<br />
m 7 GTP (μM)<br />
10 -3 10 -2 10 -1<br />
Figure 4-4. Binding isotherms for interaction <strong>of</strong> m 7 GTP <strong>with</strong> frozen (a) and fresh (b) <strong>eIF4E</strong> <strong>with</strong><br />
protein concentration fixed as 100% (broken lines, as residuals) or treated as a free parameter <strong>of</strong><br />
the fitting (solid lines, and as residuals).<br />
It is clear that the fit <strong>with</strong> the fixed protein concentration determined from<br />
absorbance before freezing <strong>of</strong> the sample ([Pact] = 0.295 μM) does not satisfy the assumed<br />
model, <strong>with</strong> R 2 = 0.2957 and P = 0.00027. The apparent association constant attains an<br />
accidental value <strong>of</strong> 18 ± 130 ⋅ 10 6 M -1 , while the fit <strong>with</strong> the active protein concentration<br />
dealt as a free parameter shows an excellent goodness <strong>of</strong> fit (R 2 = 0.9996 and P = 0.27) and<br />
yields Kas = 107.1 ± 7.8 ⋅ 10 6 M -1 but ([Pact] = 0.0266 ± 0.0014 μM. This indicates that the<br />
active fraction is only ~ 8 %! Attempts for determination <strong>of</strong> Kas in the full range <strong>of</strong> the<br />
ligand concentration <strong>with</strong> the fixed [Pact] value give still worse, worthless results.<br />
Moreover, the parameter describing the actual concentration <strong>of</strong> the active protein is crucial<br />
not only for the evidently inactivated, frozen samples, but can also change the results<br />
significantly even when the fitted curve is apparently "nice" (which is <strong>of</strong>ten the case in<br />
many literature reports, especially in those using a linear scale for ligand concentrations).<br />
Fig. 4-4(b) presents a slight difference between two fits: <strong>with</strong> [Pact] fixed as 0.2 μM and<br />
<strong>with</strong> [Pact] fitted as 0.1798 ± 0.0014 μM. Although the graphical difference between solid<br />
(R 2 = 0.9999) and broken (R 2 = 0.9990) lines is almost indistinguishable at the first sight,<br />
and the protein activity is pretty high (90%), the fit assuming the fixed [Pact] value yields a<br />
function <strong>with</strong> a systematic deviation from the experimental data, which is revealed by the<br />
residuals and the P value <strong>of</strong> < 0.0001. The latter parameter indicates that it is only less than<br />
0.01% chance that this deviation is not caused by fixing <strong>of</strong> [Pact] at the level <strong>of</strong> 100%. The<br />
Fluorescence (a. u.)<br />
Residuals<br />
54<br />
100<br />
80<br />
60<br />
40<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
2<br />
0<br />
-2<br />
m 7 GTP (μM)<br />
10 -3 10 -2 10 -1 10 0 10 1
Kas value obtained <strong>with</strong> free [Pact] is 103.2 ± 5.18 ⋅ 10 6 M -1 . Neglect <strong>of</strong> this small, 10%<br />
difference in the protein activity upon non-linear fitting <strong>with</strong> fixed [Pact] results in as many<br />
as 2-fold greater Kas <strong>of</strong> 188 ± 26 ⋅ 10 6 M -1 . Hence, the protein activity must be controlled<br />
upon each individual titration. This is especially important for strongly binding ligands,<br />
such as m 7 GTP, p-Cl-bz 7 GTP or m 7 Gppppm 7 G (Fig. 4-5). The temperatures at which the<br />
activity <strong>of</strong> <strong>eIF4E</strong> drops by 50% (34 – 41 °C) are in the physiological range, as estimated<br />
from the Boltzmann sigmoidal curves. These results could suggest that the unstable <strong>eIF4E</strong><br />
protein is usually complexed <strong>with</strong> another macromolecule (<strong>mRNA</strong> 5' cap or other proteins)<br />
in the living cell to avoid inactivation in the apo state, which is consistent <strong>with</strong> the<br />
observation that most <strong>of</strong> the <strong>eIF4E</strong> population is blocked by 4E-BPs, and biological<br />
potency <strong>of</strong> <strong>eIF4E</strong> is regulated by its accessibility. 42<br />
Active protein (%)<br />
Active protein (%)<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
(a) 0 (b)<br />
0 5 10 15 20 25 30 35 40 45<br />
Temperature (°C)<br />
(c) (d)<br />
0 5 10 15 20 25 30 35 40 45<br />
Temperature (°C)<br />
Figure 4-5. Temperature dependence <strong>of</strong> the active fraction <strong>of</strong> fresh protein ( , , , ) obtained<br />
upon titration <strong>with</strong> ligands <strong>of</strong> different affinity for <strong>eIF4E</strong>. (a) m 7 GTP, Kas = 108.7 ⋅ 10 6 M -1 ; (b)<br />
m 7 Gppppm 7 G, Kas = 47.0 ⋅ 10 6 M -1 ; (c) p-Cl-bz 7 GTP, Kas = 44.6 ⋅ 10 6 M -1 , results for frozen protein<br />
at 20°C is shown for comparison (); (d) m 7 GpppC, Kas = 3.86 ⋅ 10 6 M -1 ; the Kas values at 20 °C.<br />
55<br />
Active protein (%)<br />
log(% <strong>of</strong> active protein)<br />
100<br />
50<br />
10<br />
2<br />
0<br />
0 5 10 15 20 25 30 35 40 45<br />
Temperature (°C)<br />
-10<br />
0 5 10 15 20 25 30 35 40 45<br />
Temperature (°C)
For the ligands that bind to the protein less strongly (Kas < 10 7 M -1 ), the<br />
concentration <strong>of</strong> active protein looses its numerical importance. Determination <strong>of</strong> [Pact]<br />
becomes inaccurate due to weakly pronounced curvature <strong>of</strong> the fitted function F([L]) in the<br />
ligand concentration range close to [Pact].<br />
The population <strong>of</strong> active protein is also ionic strength-dependent (Fig. 4-6). Relative<br />
stabilization is achieved at the ionic strength characteristic for the intracellular fluid (~200<br />
mM). Determination <strong>of</strong> the active fraction <strong>of</strong> protein as a function <strong>of</strong> [KCl] was necessary<br />
to obtain association and dissociation rate constants from the kinetic traces registered<br />
during stopped-flow experiments for interaction <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> m 7 GpppG. 4 Single-<br />
relaxation fits yielded overall kinetic constants and dual-relaxation fits provided evidence<br />
for the two-step character <strong>of</strong> the complex formation.<br />
Active protein (%)<br />
100<br />
50<br />
0<br />
0 100 200 300 400<br />
KCl (mM)<br />
Figure 4-6. Dependence <strong>of</strong> the average active fraction <strong>of</strong> fresh protein on KCl concentration after<br />
storage by ~6 hours at 25 °C. Results obtained from titrations <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> m 7 GTP to control the<br />
protein activity during kinetic stopped-flow experiments <strong>with</strong> use <strong>of</strong> m 7 GpppG. 4<br />
The knowledge about the actual concentration <strong>of</strong> active non-enzymatic protein is<br />
crucial for normalization <strong>of</strong> every quantity being determined experimentally, analogically<br />
as specific activity <strong>of</strong> enzymes is used. 209 In particular, long-lasting thermodynamic<br />
measurements performed by means <strong>of</strong> techniques other than emission spectroscopy require<br />
concurrent control fluorescence titrations so that the results are free from systematic errors<br />
caused by temperature-dependent protein inactivation.<br />
56
Fluorescence (a. u.)<br />
Figure 4-7. Determination <strong>of</strong> protein active fractions at 30 °C (303.2 K, , broken lines) and 15 °C<br />
(288.1 K, , solid lines). Titration curves determine both K as and [Pact] as free parameters <strong>of</strong> the<br />
fitting (Eq. 3-26, p. 34). [Pact] is graphically represented by the point where the curve for<br />
hypothetical infinite Kas would attain maximal quenching Qmax (Eq. 3-27, p. 34).<br />
Changes in the amount <strong>of</strong> active <strong>eIF4E</strong> incubated at 288 K and 303 K during a time<br />
corresponding to duration <strong>of</strong> isothermal calorimetric titration are shown in Fig. 4-7. Total<br />
<strong>eIF4E</strong> concentration was 0.2 μM at both temperatures. Beside obvious differences between<br />
the association constants, the control fluorescence experiments have shown that <strong>eIF4E</strong><br />
retained 89.7 % activity at 288 K but only 53.5 % at 303 K. Further determination <strong>of</strong> the<br />
standard molar binding enthalpies and heat capacity changes had to be based on this<br />
information.<br />
100<br />
80<br />
60<br />
40<br />
303K<br />
Qmax Q max 288K<br />
10 -3 10 -2 10-1 10 0 101 [Pact] 20<br />
303K [Pact] 288K<br />
m 7 GTP (μM)<br />
The methodology <strong>of</strong> determination <strong>of</strong> the active protein fraction was initially<br />
designed to resolve the problem <strong>of</strong> activity control <strong>of</strong> the non-enzymatic <strong>eIF4E</strong> translation<br />
factor. 4,6 However, it was further proposed to study stoichiometry <strong>of</strong> ligand binding to an<br />
enzymatic oligoprotein and proved successful in finding that a hexameric enzyme (PNP)<br />
binds only three substrate molecules per one hexamer. 210,211<br />
57
4.1.3. Incorrectness <strong>of</strong> Data Linearization in Case <strong>of</strong> Strong <strong>Interaction</strong>s<br />
Independently from skipping the fundamental problem <strong>of</strong> the protein activity, the<br />
previous reports were based mostly on linearized forms <strong>of</strong> the equilibrium equation which<br />
are not suitable for the analysis <strong>of</strong> strong interactions. The commonly used Eadie-H<strong>of</strong>stee<br />
linear transformation requires an assumption that protein concentration is negligible in<br />
comparison <strong>with</strong> ligand concentration over the entire course <strong>of</strong> the titration. 156,161-163 This is<br />
not satisfied for molecular systems <strong>of</strong> a higher affinity. When Kas ~ 10 μM -1 , saturation <strong>of</strong><br />
~ 40% occurs already for [L] [Pact], at typical [Pact] ~ 0.1 μM. This makes the<br />
transformed data non-linear, even after corrections for dilution, inner filter effect, and free<br />
ligand emission. Additionally, the experimental uncertainty is hidden in the x-axis (Fig. 4-<br />
8). Inverse representation <strong>of</strong> the data reveals the huge errors which are always neglected<br />
during the Eadie-H<strong>of</strong>stee analysis.<br />
ΔΔF (a. u.)<br />
400<br />
300<br />
200<br />
100<br />
0<br />
2500 5000 7500 10000<br />
ΔF/[L] (a. u./μM)<br />
Figure 4-8. (a) Eadie-H<strong>of</strong>stee transformation <strong>of</strong> experimental data from the same fluorescence<br />
titration as in Fig. 4-4(a), apparent Kas = 39 ± 12 ⋅ 10 6 M -1 and (b) inverse Eadie-H<strong>of</strong>stee<br />
transformation <strong>of</strong> these data, apparent Kas = 17.2 ± 5.0 ⋅ 10 6 M -1 .<br />
A modified linear Eadie-H<strong>of</strong>stee representation 5 requires the active protein<br />
concentration and the maximal quenching (ΔFmax) as known constants (Fig. 4-9). Even<br />
though they are guessed properly, the most important data points, which should determine<br />
the binding constant, are compressed by the transformation into a very narrow numerical<br />
range (the stronger binding the more narrow the range). This leads to loss <strong>of</strong> their<br />
numerical importance, and consequently, to significantly biased Kas values.<br />
ΔF/[L] (a. u./ μM)<br />
58<br />
15000<br />
10000<br />
5000<br />
0<br />
-5000<br />
-10000<br />
0 100 200 300<br />
ΔF (a. u.)
[m 7 GTP]/ΔF (μM/a. u.)<br />
0.0006<br />
0.0004<br />
0.0002<br />
0.0000<br />
0.00 0.01 0.02 0.03<br />
1/(ΔF max-ΔF) (1/a. u.)<br />
Figure 4-9. Modified linear Eadie-H<strong>of</strong>stee representation 5 <strong>of</strong> the data from Fig. 4-4(a) and Fig. 4-8.<br />
Although the required constant parameters ([Pact] and ΔFmax) <strong>of</strong> the linear data transformation are<br />
taken from the previous fit <strong>with</strong> the free active protein concentration, shown in Fig. 4-4(a), the<br />
apparent Kas value is significantly decreased, 72.3 ± 1.2 ⋅ 10 6 M -1 , due to contraction <strong>of</strong> the most<br />
important data at the low concentrations <strong>of</strong> the ligand to a very narrow range <strong>of</strong> the x variable.<br />
The non-linear analysis is free from the systematic errors caused by the linear<br />
transformations. The methodological improvements afford possibilities for a reliable<br />
comparison <strong>of</strong> the results for the protein from different purification batches, previously<br />
frozen or not, from experiments at different temperatures, ionic strengths, and pH. The Kas<br />
values up to 10 8 M -1 , accompanied by the systematic and self-consistent structure-affinity<br />
relationship, provide an exact, quantitative test for the proper fold <strong>of</strong> the renatured protein.<br />
This assures for the first time that the equilibrium association constants reflect the true,<br />
intrinsic affinity <strong>of</strong> <strong>eIF4E</strong> for <strong>mRNA</strong> 5' cap.<br />
59
4.2. Molecular Mechanism <strong>of</strong> Recognition <strong>of</strong> <strong>mRNA</strong> 5' Cap<br />
Structure by <strong>eIF4E</strong> Cap-Binding <strong>Protein</strong><br />
4.2.1. Affinity <strong>of</strong> Cap Analogues for <strong>eIF4E</strong><br />
4.2.1.1. Equilibrium Binding Constants<br />
Kas values determined for a wide class <strong>of</strong> cap analogues (Table 4-2) vary from 10 8 M -<br />
1 for specific 7-substituted cap analogues to 10 2 M -1 for 7-unsubstituted ligands (Scheme 2-<br />
1). The human and murine proteins have the same binding affinities for <strong>eIF4E</strong>. The murine<br />
protein was used for the further studies because <strong>of</strong> technical matters, i. e. more efficient<br />
expression and purification. However, thanks to almost 100% identity, the conclusions<br />
drawn from the data are common for both proteins.<br />
The substantial differences <strong>of</strong> the Kas values follow structural modifications <strong>of</strong> cap,<br />
m 7 , 2 , 2<br />
e.g. a 760-fold reduction <strong>of</strong> Kas for 3 GTP and up to a 5000-fold drop <strong>of</strong> Kas for GTP<br />
as compared to m 7 GTP. The presence <strong>of</strong> any N(7)-substituent enhances binding, but the<br />
methyl substituent appears to be optimal. Among several elements important for specific<br />
<strong>eIF4E</strong>-cap binding, the negative electrostatic charge <strong>of</strong> the phosphate chain, which depends<br />
both on the number <strong>of</strong> phosphate groups and the presence <strong>of</strong> a second nucleotide, is <strong>of</strong><br />
primary importance. The decrease <strong>of</strong> Kas <strong>with</strong> single step-wise reduction <strong>of</strong> the phosphate<br />
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
GMP<br />
GDP<br />
GTP<br />
m 7 GMP<br />
m 7 GDP<br />
m 7 GTP<br />
10 -3 10 -2 10 -1 10 0 10 1 10 2<br />
20<br />
Concentration <strong>of</strong> cap analogue (μM)<br />
Figure 4-10. Comparison <strong>of</strong> binding isotherms for <strong>eIF4E</strong> complexes <strong>with</strong> unmethylated and<br />
methylated at N(7) guanosine mono-, di-, and triphosphates, at 20°C, pH 7.2, 100 mM KCl.<br />
60
Table 4-2. Binding free energies (ΔG°) and equilibrium association constants (Kas) for complexes <strong>of</strong><br />
murine <strong>eIF4E</strong> (28-217) and human <strong>eIF4E</strong> (1-217) <strong>with</strong> various cap analogues, at 20°C, 50 mM<br />
Hepes/KOH pH 7.2, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA.<br />
Cap-analogue Murine <strong>eIF4E</strong> (28-217) Human <strong>eIF4E</strong> (1-217)<br />
ΔG° (kJ/mol) Kas ⋅ 10 -6 (M -1 ) Kas ⋅ 10 -6 (M -1 )<br />
m 7 GTP -45.099 ± 0.088 108.7 ± 4.0 97 ± 30 1.17 ± 0.12 a<br />
m 7 GDP -41.02 ± 0.18 20.4 ± 1.5<br />
m 7 GMP -33.15 ± 0.20 0.806 ± 0.067<br />
m 7 G -20.54 ± 0.63 0.0046 ± 0.0012<br />
et 7 GTP -39.38 ± 0.20 10.41 ± 0.86<br />
bz 7 GTP -40.660 ± 0.059 17.59 ± 0.43<br />
p-Cl-bz 7 GTP -42.93 ± 0.21 44.6 ± 3.8<br />
m 7 , 2<br />
2 GTP<br />
-41.89 ± 0.20 29.2 ± 2.4<br />
m 7 , 2 , 2<br />
3 GTP -28.9 ± 1.4 0.143 ± 0.080<br />
m 7 Gpppm 2’O G -38.75 ± 0.16 8.04 ± 0.51<br />
m 7 GpppG -38.55 ± 0.15 7.39 ± 0.46 9.6 ± 0.8 0.69 ± 0.07 a<br />
m 7 GpppA -37.43 ± 0.27 4.68 ± 0.52<br />
m 7 GpppC -36.41 ± 0.40 3.08 ± 0.51 4.8 ± 0.4 0.48 ± 0.05 a<br />
m 7 Gpppm 7 (micro) b<br />
G -35.28 ± 0.36 1.93 ± 0.28<br />
m 7 Gpppm 7 (macro) b<br />
G 3.86 ± 0.56<br />
m 7 GppppG -44.96 ± 0.11 102.8 ± 4.4<br />
m 7 Gppppm 7 (micro) b<br />
G -41.37 ± 0.15 23.5 ± 1.4<br />
m 7 Gppppm 7 (macro) b<br />
G 47.0 ± 2.7<br />
GTP -24.30 ± 0.11 0.0214 ± 0.0010<br />
GDP -20.71 ± 0.25 0.0049 ± 0.0005<br />
GMP -12.93 ± 3.8 0.0002 ± 0.0003<br />
(micro) b<br />
GpppG -20.66 ± 0.21 0.0048 ± 0.0004<br />
(macro) b<br />
GpppG 0.0095 ± 0.0007<br />
a values for m 7 G-affinity purified full length human <strong>eIF4E</strong> 5<br />
b microscopic and macroscopic association constants, see text for details.<br />
chain in the series <strong>of</strong> 7-methylated mononucleotides is followed by the same changes <strong>of</strong><br />
Kas for the unmethylated compounds (~5- and ~25-fold for removal <strong>of</strong> the γ-phosphate and<br />
the β-phosphate, respectively). Removal <strong>of</strong> the α-phosphate leads to a drastic fall <strong>of</strong> Kas,<br />
which is 175-fold lower for m 7 G than that <strong>of</strong> m 7 GMP (Fig. 4-10).<br />
61
The dinucleotide triphosphate cap analogues also bind to <strong>eIF4E</strong> less strongly (~20-<br />
fold) than m 7 GTP. The identity <strong>of</strong> the second base seems to be <strong>of</strong> minor importance.<br />
Generally, either base <strong>of</strong> the dinucleotide cap analogues penetrate the binding slot <strong>of</strong><br />
<strong>eIF4E</strong>, but since the association constants <strong>of</strong> unmethylated molecules are far lower than<br />
those <strong>of</strong> the methylated ones, such dual binding is negligible for asymmetrical cap<br />
analogues. By contrast, for symmetrical molecules (m 7 Gpppm 7 G, m 7 Gppppm 7 G and<br />
GpppG) the observed Kas must be divided by 2 because <strong>of</strong> entropic effects. Only this<br />
normalized Kas represents a true microscopic association constant Kas (micro) , reflecting the<br />
intrinsic stabilization energy <strong>of</strong> the complex. The macroscopic Kas, however, corresponds<br />
to the effective biological potency <strong>of</strong> the cap analogues.<br />
In the pioneering work <strong>of</strong> Carberry et al., 156 the association constants <strong>of</strong> two<br />
fundamental cap analogues, m 7 GTP and m 7 GpppG, for <strong>eIF4E</strong> from human erythrocytes<br />
purified by cap-affinity chromatography were determined as 3.87 ⋅ 10 5 M -1 and 3.70 ⋅ 10 5<br />
M -1 , respectively (23°C, pH 7.6, <strong>with</strong>out KCl in the buffer). Similar Kas values in the range<br />
<strong>of</strong> 10 5 -10 6 M -1 were reported for recombinant human <strong>eIF4E</strong> purified by the same<br />
method. 5,161-163 In those studies also no essential difference in affinity between m 7 GTP and<br />
m 7 GpppG was observed. While the association constants reported here for the murine<br />
protein are at variance <strong>with</strong> these data, they are very close to those obtained for<br />
recombinant full length human <strong>eIF4E</strong>, which was also purified via refolding from inclusion<br />
bodies <strong>with</strong>out any prior contact <strong>with</strong> cap (Table 4-2). These Kas values are up to 500-fold<br />
higher than those reported previously.<br />
4.2.1.2. Binding Affinity vs Inhibitory Properties <strong>of</strong> Cap Analogues<br />
The affinities represented by the present Kas values (Table 4-2) reveal large<br />
differences among cap analogues possessing different functional groups and correspond<br />
well to the inhibitory properties <strong>of</strong> the cap analogues observed in a rabbit reticulocyte<br />
lysate (Fig. 4-11). In a kinetic model developed for the in vitro translational<br />
system, 157,212,213 the inhibition constant (KI) was determined as the overall dissociation<br />
constant <strong>of</strong> the cap analogue from the 48S initiation complex. Results <strong>of</strong> both approaches,<br />
biophysical and biochemical, point out the significance <strong>of</strong> the same structural features <strong>of</strong><br />
the cap responsible for specific recognition, e.g. the presence and type <strong>of</strong> N(7)-substituent<br />
<strong>of</strong> guanine, the negative charge density in the phosphate chain, and the N 2 amino group<br />
<strong>with</strong> at least one proton capable <strong>of</strong> forming a hydrogen bond. Interestingly, the observed<br />
62
values <strong>of</strong> KI are ~100 times greater than those <strong>of</strong> 1/Kas for all cap analogues. This<br />
apparently larger amount <strong>of</strong> cap analogue required to inhibit translation in vitro than that<br />
necessary to displace <strong>eIF4E</strong> from the <strong>mRNA</strong> cap most likely arises from the following<br />
reasons. As opposed to HeLa cells, 41 the <strong>eIF4E</strong> concentration in reticulocyte lysate is not<br />
limiting for translation, and the majority <strong>of</strong> <strong>eIF4E</strong> is not engaged in this process. 214 The<br />
assumption underlying the determination <strong>of</strong> KI values, that <strong>eIF4E</strong> concentration is<br />
negligible in comparison <strong>with</strong> that <strong>of</strong> a competitive inhibitor, 213 is not applicable here. The<br />
concentration <strong>of</strong> <strong>eIF4E</strong> is up to 50-fold higher than previously thought, 215 so much more<br />
cap analogue is required to inhibit translation due to the quadratic form <strong>of</strong> the equilibrium<br />
equation. Besides, protein synthesis results from a large number <strong>of</strong> catalytic reactions and<br />
depends not only on the <strong>eIF4E</strong> activity, e. g. in the in vitro experiments, the <strong>mRNA</strong><br />
binding activity <strong>of</strong> eIF4F complex is enhanced by the presence <strong>of</strong> eIF4G. 216<br />
KI [μμM]<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
10 -2 10 -1 10 0 10 1 10 2<br />
K as [μM -1 ]<br />
Figure 4-11. Correlation between inhibition constants <strong>of</strong> the cap analogues (KI), obtained from in<br />
vitro translation (30°C 157 ), and their equilibrium association constants (Kas, 20°C; macroscopic Kas<br />
were used for the symmetrical caps) by 4 orders <strong>of</strong> magnitude. Correlation coefficient r 2 = 0.795.<br />
63
4.2.2. Parsing the Free Energy <strong>of</strong> <strong>eIF4E</strong> Binding to <strong>mRNA</strong> 5' Cap<br />
A comparison <strong>of</strong> the binding free energies rather than that <strong>of</strong> the association<br />
constants reflects the biophysical basis <strong>of</strong> the ligand affinity for the protein. The data<br />
collected for twenty structurally different cap analogues permitted analysis <strong>of</strong> the influence<br />
<strong>of</strong> single structural modifications on the cap-<strong>eIF4E</strong> binding in terms <strong>of</strong> the standard Gibbs<br />
free energies ΔG° (Table 4-2), which were calculated either from the binding constants Kas,<br />
Δ G° = −RT<br />
ln K as , or from Kas (micro) ,<br />
64<br />
( micro)<br />
as<br />
Δ G° = −RT<br />
ln K , for the symmetrical cap<br />
analogues. Contribution <strong>of</strong> a given chemical alteration is represented by the corresponding<br />
change <strong>of</strong> the standard Gibbs energy <strong>of</strong> association (ΔΔG°), defined individually for each<br />
group <strong>of</strong> compounds in Table 4-3.<br />
It should be noted here that parsing is generally not applicable for every molecular<br />
system. 152,153 However, direct comparison <strong>of</strong> the ΔG° values surprisingly but distinctly<br />
showed that the total binding free energy resolved itself into individual contributions from<br />
interactions <strong>of</strong> the phosphate chain and into common contribution from all interactions <strong>of</strong><br />
the m 7 guanosine moiety. This limited additivity is a kind <strong>of</strong> empirical verification <strong>of</strong><br />
correctness <strong>of</strong> parsing employment in this particular case, and, on the other hand, points to<br />
cooperativity <strong>of</strong> interactions involving the m 7 guanosine moiety (stacking and hydrogen<br />
bonding) that may not be decomposed into their contributions.
Table 4-3. Changes in the standard Gibbs free energy (ΔΔG°) on <strong>eIF4E</strong> binding to the<br />
structurally modified cap analogues, at 20°C.<br />
Structural alteration ΔΔG° ΔΔ kJ/mol<br />
replacement <strong>of</strong> N(7)-methyl for larger substituents:<br />
ΔΔG° = ΔG°(x 7 GTP) − ΔG°(m 7 GTP)<br />
m → x : m → et m → bz m → p-Cl-bz<br />
x 7 GTP +5.73 ± 0.21 +4.44 ± 0.13 +2.18 ± 0.21<br />
methylation at N(7) for n phosphate groups:<br />
ΔΔG° = ΔG°(m 7 Gpn(G)) − ΔG°(Gpn(G))<br />
n: 1 2 3<br />
Gpn → m 7 Gpn −20.2 ± 3.8 −20.29 ± 0.29 −20.79 ± 0.13<br />
GpnG a → m 7 GpnG −17.91 ± 0.25<br />
addition or alteration <strong>of</strong> the second nucleoside:<br />
ΔΔG° = ΔG°(m 7 GpnY) − ΔG°(m 7 GpnX)<br />
X → Y: none → G G → m 7 G a<br />
G → A G → C G → m 2’O G<br />
m 7 Gp3X → m 7 Gp3Y +6.57 ± 0.17 +3.26 ± 0.38 +1.13 ± 0.29 +2.13 ± 0.42 −0.2 ± 0.2<br />
m 7 Gp4X → m 7 Gp4Y +3.60 ± 0.17<br />
Gp3X → Gp3Y a<br />
+3.64 ± 0.25<br />
successive addition <strong>of</strong> the 5'-phosphate groups:<br />
ΔΔG° = ΔG°(n+1) − ΔG°(n)<br />
n → n+1: 0 → 1 1 → 2 2 → 3 3 → 4<br />
m 7 Gpn −12.59 ± 0.67 −7.87 ± 0.25 −4.10 ± 0.21<br />
Gpn −7.8 ± 3.8 −3.60 ± 0.29<br />
m 7 GpnG −6.40 ± 0.17<br />
m 7 Gpnm 7 G a<br />
−6.11 ± 0.38<br />
successive addition <strong>of</strong> the N 2 -methyl groups:<br />
ΔΔG° = ΔG°(n+1) − ΔG°(n)<br />
n → n+1: 0 → 1 1 → 2<br />
2,<br />
2 7<br />
m n m GTP +0.77 ± 0.05 +3.10 ± 0.33<br />
a for symmetrical cap analogues ΔΔG° calculated from the microscopic association constant Kas (micro)<br />
65
4.2.2.1. Energetic Cost <strong>of</strong> 7-Substituent Alteration<br />
The presence <strong>of</strong> each type <strong>of</strong> substituent at the N(7) position <strong>of</strong> the guanine moiety<br />
produces a comparable effect on the <strong>eIF4E</strong> binding enhancement (Fig. 4-12), in<br />
comparison <strong>with</strong> the non-substituted cap analogue, by ΔΔG° <strong>of</strong> −15 to −21 kJ/mol (Tables<br />
4-2, 4-3). The main gain <strong>of</strong> the stacking energy is reached irrespective <strong>of</strong> the nature <strong>of</strong> the<br />
substituent. This indicates that the delocalized positive charge resulting from N(7)-<br />
substitution is <strong>of</strong> primary importance. The only direct interaction involving the 7-methyl<br />
group is a non-specific van der Waals contact <strong>with</strong> Trp166 in the complex <strong>with</strong> m 7 GDP<br />
(3.93 Å, Fig. 4-13a), which is lost in the complex <strong>with</strong> m 7 GpppG (4.17 Å, Fig. 4-13b).<br />
Replacement <strong>of</strong> the methyl group for larger substituent (ethyl, benzyl) can cause steric<br />
hindrance, and can interfere <strong>with</strong> creation <strong>of</strong> the water-mediated hydrogen bonds<br />
stabilizing the α-phosphate. Thus, it is energetically unfavourable by ΔΔG° <strong>of</strong> +2.2 to +5.7<br />
kJ/mol.<br />
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
et 7 GTP<br />
p-Cl-bz 7 GTP<br />
m 7 GTP<br />
bz 7 GTP<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
Concentration <strong>of</strong> cap analogue (μM)<br />
Figure 4-12. Binding isotherms for <strong>eIF4E</strong> complexes <strong>with</strong> N(7)-substituted guanosine<br />
triphosphates, at 20°C, pH 7.2, 100 mM KCl.<br />
66
Trp-166<br />
Trp-166<br />
Glu-103<br />
Trp-102<br />
Glu-103<br />
Trp-102<br />
Arg-112<br />
Arg-112<br />
c<br />
n2<br />
Figure 4-13. Interatomic contacts in (a) m 7 GDP-<strong>eIF4E</strong> complex, determined on the basis <strong>of</strong> the<br />
crystallographic structure (PDB ID code: 1EJ1), 75 and (b) m 7 GpppG-<strong>eIF4E</strong> complex. Hydrogen<br />
bonds, salt bridges and van der Waals interactions are indicated <strong>with</strong> dotted lines, <strong>with</strong> the lengths<br />
expressed in Å. Trapped water molecules supporting the hydrogen-bonded network are shown as<br />
magenta spheres. The fragments <strong>of</strong> <strong>eIF4E</strong> distant from the cap by 4 Å are shown as spheres.<br />
67<br />
Lys-162<br />
n2<br />
Lys-162<br />
Lys-206<br />
Trp-56<br />
c5'<br />
Trp-56<br />
c5'<br />
(a)<br />
Arg-157<br />
(b)<br />
Arg-157
4.2.2.2. General Separation <strong>of</strong> the Binding Free Energy<br />
The binding free energy <strong>of</strong> the 7-methylated analogues association <strong>with</strong> <strong>eIF4E</strong><br />
appears to be totally separated into two groups <strong>of</strong> interactions: stacking together <strong>with</strong><br />
hydrogen bonding and binding through the phosphates. Irrespective <strong>of</strong> the level <strong>of</strong> the cap<br />
analogue phosphorylation, methylation at N(7) is accompanied by almost constant binding<br />
free energy change, ΔΔG° about –20.5 kJ/mol (Table 4-3). The gain from N(7)-<br />
methylation slightly decreases for the shorter phosphate chains. This minute effect may<br />
result from the fact that acidic-basic properties <strong>of</strong> N(1)-H proton slightly depend on the<br />
number <strong>of</strong> the phosphate groups 177 (Scheme 3-1, p. 23). Therefore, the m 7 G moiety<br />
assumes the required cationic form (see 4.2.3.2., p. 83) more readily in analogues <strong>with</strong> a<br />
longer phosphate chain. However, both the pKa-shifts 177 and differences among ΔΔG°<br />
values related to methylation <strong>of</strong> mono-, di- and triphosphates (Table 4-3) are very small,<br />
indicating that the presence <strong>of</strong> the phosphates influences the charge <strong>of</strong> m 7 G to a negligible<br />
extent. The charge flow via the ribose σ-bonds does not occur.<br />
Surprisingly, the measured values <strong>of</strong> ΔG° satisfy the relationship: ΔG°(m 7 Gpn) =<br />
ΔG°(m 7 G) + ΔG°(G pn), for n = 1, 2 or 3. This means that the 7-methylguanosine binding<br />
is separate from binding <strong>of</strong> the phosphate chain. The possible entropic cost arising from a<br />
change in the degrees <strong>of</strong> freedom due to linkage <strong>of</strong> the two moieties is either negligible or<br />
can be partially cancelled out if the bound phosphates facilitate to some extent penetration<br />
<strong>of</strong> the cap-binding slot by m 7 G. The resultant entropic effect is, however, below the best<br />
experimental accuracy: δ(ΔG°(m 7 Gpn) − ΔG°(m 7 G) − ΔG°(Gpn)) = ± 0.67 kJ/mol. The<br />
total binding free energy <strong>of</strong> 7-methylguanosine, ΔG°(m 7 G) = −20.54 ± 0.63 kJ/mol (Table<br />
4-2), is the same as ΔΔG° resulting from methylation at N(7), which demonstrates that the<br />
ribose does not contribute to the complex stabilization. The 3D structures show only one<br />
non-specific van der Waals contact between the ribose and Trp56 (Fig. 4-13).<br />
The stabilization energy <strong>of</strong> the GMP-<strong>eIF4E</strong>, GDP-<strong>eIF4E</strong> and GTP-<strong>eIF4E</strong> complexes<br />
originates only from the interactions involving the phosphate groups, independently<br />
pointing to the minute role for the ribose in stabilization <strong>of</strong> the complex. It is directly seen<br />
from the comparison <strong>of</strong> the total binding energy, ΔG°(GMP) = −12.93 ± 3.8 kJ/mol (Table<br />
4-2), <strong>with</strong> the contribution <strong>of</strong> the single α-phosphate, ΔΔG° = −12.59 ± 0.67 kJ/mol (Table<br />
4-3). These findings reveal that although unmethylated guanosine phosphates are<br />
potentially capable to form three Watson-Crick like hydrogen bonds, such bonds are not<br />
68
created. It is only when the guanine moiety possesses a substituent at the N(7) position that<br />
the hydrogen bonds are formed. As shown above, the positive charge at m 7 G determines<br />
the cap affinity for <strong>eIF4E</strong> being a cause <strong>of</strong> the stacking enhancement.<br />
The most striking conclusion is that the cation-π stacking enhancement is a<br />
precondition for the hydrogen bond formation, and a double role should be attributed to the<br />
presence <strong>of</strong> the conserved tryptophans 56 and 102 in the <strong>eIF4E</strong> binding site: stabilization<br />
<strong>of</strong> the 7-methylguanine ring by stacking itself and enabling 7-methylguanine to form the<br />
hydrogen bonds <strong>with</strong> the carboxylate oxygen atoms <strong>of</strong> Glu-103 and the backbone amino<br />
group <strong>of</strong> Trp-102 (Fig. 4-13). Consequently, the system <strong>of</strong> m 7 G-<strong>eIF4E</strong> works according to<br />
the "all-or-nothing" rule.<br />
4.2.2.3. Relation <strong>of</strong> Stacking - Hydrogen Bonding Cooperativity to Other Cap-<br />
Binding Molecules<br />
Previous studies using small model peptides showed that the stacking interactions <strong>of</strong><br />
N(7)-methylated guanine <strong>with</strong> the aromatic rings <strong>of</strong> Trp, Tyr, Phe are stronger in<br />
comparison <strong>with</strong> the unmethylated base. However, the difference in the Kas values was<br />
lesser than 1.5-fold. 217 On the other hand, spectroscopic studies on the interaction between<br />
cap analogues and small model peptides, containing Trp and Glu, showed simultaneous<br />
formation <strong>of</strong> hydrogen bonds between 7-methylguanine and the Glu carboxyl side group,<br />
and stacking <strong>with</strong> the Trp indole ring. 218-220 These investigations alone, however, do not<br />
explain why compounds containing unmethylated guanine moiety, capable to form three<br />
hydrogen bonds involving O 6 , N(1) and N 2 , do not competitively inhibit translation 157 and<br />
are unable to bind <strong>eIF4E</strong> as well as an other cap-binding protein, the <strong>mRNA</strong> 5' cap-specific<br />
viral methyltransferase VP39. 221,222 Enhanced stacking <strong>of</strong> the methylated, positively<br />
charged bases <strong>with</strong> two parallel aromatic side chains <strong>of</strong> VP39 (Tyr22 and Phe180) plays a<br />
dominant role in the cap-recognition, 221 and hydrogen bonding is <strong>of</strong> secondary<br />
importance. 223 The single- and double-substitutions <strong>of</strong> Tyr22 and Phe180 by more strongly<br />
stacking tryptophans were shown to be associated <strong>with</strong> increased affinity for 7-<br />
methylguanosine by factors <strong>of</strong> 10 and 50, respectively. 224 The structural requirements for<br />
the specific cap recognition were qualitatively examined by means <strong>of</strong> the fluorometric<br />
titration applied to GST-fused <strong>eIF4E</strong> and His-tagged VP39, <strong>with</strong> amino acid substitutions<br />
in the cap-binding sites <strong>of</strong> the proteins. 164 The results suggest that, in order to permit the<br />
selective binding <strong>of</strong> m 7 GDP, <strong>eIF4E</strong> and VP39 require both the aromatic residues and a<br />
69
glutamic acid in a specific mutual orientation. Although VP39 and <strong>eIF4E</strong> do not share a<br />
common phylogenetical ancestor, they use a similar mode <strong>of</strong> cap binding.<br />
Application <strong>of</strong> the exact quantitative approach makes it possible now to elucidate this<br />
strict stacking/hydrogen bonding cooperativity. The observed entirely different affinity <strong>of</strong><br />
the 7-methylated nucleotides as compared <strong>with</strong> their unmethylated counterparts does not<br />
arise from different acidic/basic properties <strong>of</strong> the two classes <strong>of</strong> compounds, as was<br />
suggested previously, 156 but originates from their different stacking ability.<br />
4.2.2.4. Energetic Cost <strong>of</strong> Methylation <strong>of</strong> the N(2)-Amino Group<br />
Large ΔΔG° <strong>of</strong> +13 kJ/mol that accompanies substitution <strong>of</strong> the second amino-proton<br />
m 7 , 2 , 2<br />
for the methyl group at N 2 <strong>of</strong> guanine in 3 GTP points to disruption <strong>of</strong> possibly more<br />
than one hydrogen bond between <strong>eIF4E</strong> and the cap. If one subtracts the steric and<br />
hydrophobic costs (ΔΔG° <strong>of</strong> about +3 kJ/mol, as observed for the methyl substitution <strong>of</strong><br />
m 7 , 2<br />
2<br />
the first amino-proton in GTP , Table 4-3), the resulting energy (about +10 kJ/mol)<br />
suggests breaking <strong>of</strong> not only the Glu103–COO...HN 2 –m 7 GTP hydrogen bond but also the<br />
second hydrogen bond, Glu103–COO...HN(1)–m 7 GTP, most likely due to steric effects <strong>of</strong><br />
the bulk dimethylamino group, which can move the acceptor group <strong>of</strong> Glu103 away.<br />
4.2.2.5. Energetic Cost <strong>of</strong> the Second Nucleoside Addition and Modification<br />
All <strong>of</strong> the dinucleoside triphosphates bind to <strong>eIF4E</strong> less strongly than m 7 GTP, by<br />
ΔΔG° <strong>of</strong> +6.6 to +10 kJ/mol, and even <strong>with</strong> lesser strength than m 7 GDP (Tables 4-2, 4-3).<br />
The decreased affinity suggests that the second nucleoside does not bind tightly to <strong>eIF4E</strong>.<br />
Independently from the affinity studies, the same conclusion arises from the cocrystal<br />
structure <strong>of</strong> m 7 GpppG bound to <strong>eIF4E</strong>. Figures 4-13b and 4-14 show the only part <strong>of</strong><br />
m 7 GpppG, which can be detected from the crystallographic data. There is no defined<br />
electron density for the second guanine nucleoside. 1<br />
The presence <strong>of</strong> the unbound fragment <strong>of</strong> the cap analogue can destabilize the<br />
remaining intermolecular contacts. Addition <strong>of</strong> the second guanosine to GTP gives<br />
disadvantageous entropic effect by ΔΔG° <strong>of</strong> about +3.6 kJ/mol, while the second<br />
guanosine attached to m 7 GTP results in ΔΔG° about +6.6 kJ/mol (Table 4-3). This larger<br />
value, together <strong>with</strong> the former finding that GTP is bound only through the phosphates (see<br />
4.2.2.2., p.68), can indicate that the interactions <strong>of</strong> the 7-methylguanine moiety in<br />
70
Figure 4-14. Stereo view <strong>of</strong> the geometry <strong>of</strong> the <strong>eIF4E</strong> cap-binding site <strong>with</strong> the only part <strong>of</strong><br />
m 7 GpppG, which was detected by crystallography. 1 Molecular electrostatic potential <strong>of</strong> the <strong>eIF4E</strong><br />
surface at pH 6.0 in the absence <strong>of</strong> the ligand was calculated by <strong>Protein</strong> Explorer 225 (red and blue<br />
represent negatively and positively charged regions, respectively). The ligand is represented as<br />
sticks (carbon – green, other atoms – CPK colours). Water molecules are shown as magenta balls.<br />
m 7 GpppG are also disturbed by the unbound second nucleoside "hanging" on the<br />
phosphate chain, by ΔΔG° <strong>of</strong> about +3 kJ/mol. The crystallographic structure <strong>of</strong> the<br />
m 7 GpppG-<strong>eIF4E</strong> complex shows clearly, that the hydrogen bonds <strong>with</strong> Glu103 (donor-<br />
acceptor distances > 3Å) and the van der Waals interaction <strong>of</strong> the 7-methyl group <strong>with</strong><br />
Trp166 (distance > 4Å) are destabilized (Fig. 4-13b).<br />
In the other crystal complex <strong>of</strong> m 7 GpppA <strong>with</strong> full length human <strong>eIF4E</strong>, 48 the second<br />
adenosine is visible in the electron density map. It is stabilized by a direct hydrogen bond<br />
between adenosine N 6 -amino group and the backbone carbonyl <strong>of</strong> Thr205. Guanosine<br />
possesses a hydrogen acceptor at C(6), which precludes formation <strong>of</strong> this bond, and the<br />
protein loop containing Thr205 is disordered. The binding free energy (ΔG°) <strong>of</strong> m 7 GpppA<br />
is slightly less than that <strong>of</strong> m 7 GpppG (Table 4-2), which was recently confirmed by<br />
subsequent studies. 74 Hence, the energetic gain from the additional stabilization contact <strong>of</strong><br />
the adenosine does not compensate for the entropic cost related to the ordering <strong>of</strong> the C-<br />
terminal loop.<br />
71
Methylation <strong>of</strong> the second guanine at N(7) decreases the affinity <strong>of</strong> tri- and<br />
tetraphosphate dinucleotide caps to the same extent, ΔΔG° about +3.4 kJ/mol, consistent<br />
<strong>with</strong> attenuation <strong>of</strong> the negative charge <strong>of</strong> the phosphate chain (see below) and, at least<br />
partially, <strong>with</strong> the entropic cost <strong>of</strong> ordering the larger molecule. Exchange <strong>of</strong> the second<br />
base for adenine or cytosine is energetically less favourable. A minute advantageous effect<br />
in comparison <strong>with</strong> m 7 GpppG appears after methylation <strong>of</strong> the second ribose at O(2'),<br />
which is however in the range <strong>of</strong> the experimental error.<br />
4.2.2.6. Contributions <strong>of</strong> Phosphate Groups and Trapped Water Molecules<br />
Data in Table 4-3 show a clear accordance <strong>of</strong> the ΔΔG° values related to the<br />
phosphate interactions between two series <strong>of</strong> compounds: 7-methylated and nonmethylated<br />
guanosine phosphates. Addition <strong>of</strong> the phosphate groups one after another is accompanied<br />
by formation <strong>of</strong> direct or water-mediated hydrogen bonds and salt bridges, yielding the<br />
same free energies in the two groups <strong>of</strong> compounds. This finding shows again that there is<br />
no influence <strong>of</strong> the positive charge resulting from N(7)-methylation on the charge<br />
distribution at the phosphate chain. ΔΔG° for each step agrees <strong>with</strong> the number <strong>of</strong><br />
intermolecular contacts revealed by the crystallographic structures <strong>of</strong> the <strong>eIF4E</strong>-cap<br />
complexes (Fig. 4-13). The α-phosphate forms a direct salt bridge <strong>with</strong> Nχ2 <strong>of</strong> Arg157 as<br />
well as three indirect hydrogen bonds <strong>with</strong> Nε1 <strong>of</strong> Trp102, Nε1 <strong>of</strong> Trp166 and Nχ2 <strong>of</strong><br />
Arg112 via five water molecules trapped in the cavity <strong>of</strong> the binding centre. All water-<br />
mediated hydrogen bonds satisfy a requirement <strong>of</strong> tetrahedral geometry <strong>of</strong> the water<br />
molecules and the Pα-oxygen anion. These five bridging water molecules support the same<br />
hydrogen-bonded network both in the <strong>eIF4E</strong>-m 7 GDP complex 75 and the <strong>eIF4E</strong>-m 7 GpppG<br />
complex, and are present also in the crystallographically independent second complexes in<br />
the asymmetric units. It should be noted here that such specific network <strong>of</strong> water molecules<br />
in the protein interior is sometimes very rigid, and the water molecules buried in the<br />
internal cavities increase the protein stability. 226 Moreover, the internal water scaffold<br />
which is directly involved in ligand binding contributes to the protein specificity attained<br />
by the evolutionary specialization. 227<br />
The total free energy <strong>of</strong> the α-phosphate stabilization is ΔΔG° = −12.59 ± 0.67<br />
kJ/mol (Table 4-3). The β-phosphate group makes a salt bridge <strong>with</strong> Nζ <strong>of</strong> Lys162 and a<br />
hydrogen bond <strong>with</strong> Nε <strong>of</strong> Arg157, <strong>with</strong> the total ΔΔG° = −7.87 ± 0.25 kJ/mol, i.e. about<br />
72
−4 kJ/mol per one direct bond. This indicates average ΔΔG° <strong>of</strong> about –3 kJ/mol per one<br />
water-mediated hydrogen bond in case <strong>of</strong> the α-phosphate. The latter value is slightly less<br />
than typical magnitudes <strong>of</strong> hydrogen bonds and salt bridges in a solvent accessible region<br />
in proteins (–5.4 ± 2.5 kJ/mol) 141-143 due to the entropic cost <strong>of</strong> ordering the water<br />
molecules. The γ-phosphate <strong>of</strong> m 7 GpppG is stabilized by an additional, bifurcated salt<br />
bridge <strong>with</strong> Nζ <strong>of</strong> Lys206 <strong>of</strong> a similar energy about –3.9 kJ/mol (Fig. 4-13b).<br />
Extension <strong>of</strong> the phosphate chain by the δ-moiety in the dinucleotide cap analogues<br />
gives an energetic gain <strong>of</strong> about −6.3 kJ/mol, what cancels out the energetic cost <strong>of</strong> the<br />
second guanosine addition to m 7 GTP (Table 4-3). Most likely the δ-phosphate does not<br />
interact directly <strong>with</strong> <strong>eIF4E</strong> but the four-membered chain secures the necessary<br />
conformational freedom for the second nucleoside which is not bound, enabling the<br />
remaining m 7 Gppp-part <strong>of</strong> the cap to bind tightly. Thus, the presence <strong>of</strong> the δ-phosphate in<br />
m 7 GppppG leads to formation <strong>of</strong> the same intermolecular contacts as for m 7 GTP (ΔG° =<br />
−45.099 ± 0.088 kJ/mol) and restores the affinity <strong>of</strong> the dinucleotide cap analogue to the<br />
same level (ΔG° = −44.96 ± 0.11 kJ/mol). However, it cannot be excluded that the δ-<br />
phosphate is additionally stabilized, and the entropic cost due to loss <strong>of</strong> some<br />
conformational degrees <strong>of</strong> freedom <strong>of</strong> the second nucleoside partially cancels the<br />
stabilization effect, thus leading to accidental agreement.<br />
4.2.3. <strong>Interaction</strong>s in Context <strong>of</strong> Environment<br />
Interpretation <strong>of</strong> structural and <strong>of</strong> thermodynamic data obtained under a single set <strong>of</strong><br />
solution conditions is complicated by the fact that ligand binding by proteins is an<br />
exchange reaction. Functional groups <strong>of</strong> both protein and the ligand are capable <strong>of</strong><br />
interacting not only <strong>with</strong> each other, but also <strong>with</strong> water and solutes. Hydrogen bonds <strong>with</strong><br />
water and Coulombic interactions <strong>of</strong> phosphates <strong>with</strong> cations may be comparable to the<br />
protein-ligand interactions that replace them in the specific complex. In order to evaluate<br />
the role ions and water upon the <strong>eIF4E</strong>-cap complex formation the dependence <strong>of</strong> the Kas<br />
values <strong>of</strong> several cap analogues on salt concentration and pH have been investigated.<br />
73
4.2.3.1. <strong>Interaction</strong> <strong>of</strong> <strong>eIF4E</strong>-Cap in the Presence <strong>of</strong> Electrolyte<br />
The process <strong>of</strong> <strong>eIF4E</strong>-cap binding has been examined regarding the salt effects in<br />
two complementary ways: according to Davies-Stockes-Robinson electrostatic screening<br />
theory 191,192 and according to stoichiometric Wyman linkage analysis. 193<br />
4.2.3.1.1. Davies-Stockes-Robinson Electrostatic Screening Approach<br />
4.2.3.1.1.1. Electrostatic Effects<br />
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
10 -3 10 -2 10 -1 10 0 10 1 10 2<br />
m 7 GppppG (μM)<br />
Figure 4-15. Exemplary <strong>eIF4E</strong> binding isotherms for m 7 GppppG at different KCl concentrations<br />
added to Hepes 50 mM, pH 7.2, at 20 °C (, 20 mM; , 100 mM; , 201 mM; , 500 mM).<br />
At elevated ionic strength (I) the interaction is strongly attenuated (Fig. 4-15, Table<br />
4-4). Screening <strong>of</strong> the electrostatic attraction between <strong>eIF4E</strong> and the cap analogues <strong>of</strong><br />
increasing negative charges, i.e. m 7 GDP, m 7 GpppG, m 7 GTP and m 7 GppppG, is<br />
qualitatively shown in Fig. 4-16, on the basis <strong>of</strong> the simple Brønstes-Bjerrum-Debye-<br />
Hückel limited law. ∗ This approximation, although too far-reaching, is useful to show a<br />
certain experimental regularity. The negative slope <strong>of</strong> the regression line is approximately<br />
proportional to the charge at the phosphate chain, assuming that the terminal phosphates in<br />
the mononucleotide cap analogues are not fully ionized at pH 7.2 (pKa phosph ~6.1-6.5,<br />
depending on the number <strong>of</strong> phosphate groups 178 ).<br />
∗ A linear dependence <strong>of</strong> logKas on the square root <strong>of</strong> ionic strength (I 1/2 ); assumptions: very low salt<br />
concentration (< 0.01 M), dissociation rate constant the same for each I value, point charges (aj = 0), no<br />
hydration (ΔN = 0). None <strong>of</strong> the assumptions is fulfilled in the case <strong>of</strong> macromolecular interactions at the salt<br />
concentration close to physiological conditions, hence no quantitative conclusions can be drawn.<br />
74
log Kas - log Kas(0)<br />
0<br />
-1<br />
-2<br />
-3<br />
-4<br />
m 7 m<br />
GppppG<br />
7 m<br />
Gppp<br />
7 Gpp<br />
m 7 GpppG<br />
0.0 0.2 0.4 0.6 0.8<br />
I 1/2 (M 1/2 )<br />
Figure 4-16. The ionic strength-dependent decrease <strong>of</strong> the relative affinity for <strong>eIF4E</strong> <strong>of</strong> four<br />
selected cap-analogues <strong>with</strong> increasing negative charges. The approximate slopes are the steepest<br />
the more negative are the charges <strong>of</strong> the phosphate chains. Kas(0) is the value <strong>of</strong> the association<br />
constant at I = 0, extrapolated on the basis <strong>of</strong> the linear regression.<br />
Table 4-4. Equilibrium association constants for binding <strong>of</strong> <strong>eIF4E</strong> to the cap analogues at 20 °C,<br />
pH 7.2, in Hepes/KOH 50 mM (pKa = 7.5), and at the concentration <strong>of</strong> added KCl as listed below.<br />
Ionic strength was calculated taking into account contribution from partially ionized buffer. The<br />
slopes and the correlation coefficients (r 2 ) have been obtained by linear regression based on the<br />
Brønstes-Bjerrum-Debye-Hückel limited law.<br />
[KCl] m 7 GDP m 7 GpppG m 7 GTP m 7 GppppG<br />
(mM) Kas ⋅ 10 -6 (M -1 )<br />
5.3 1900 ± 3200<br />
20 40.7 ± 5.6 34.4 ± 3.8 1000 ± 460 1112 ± 530<br />
50 17.9 ± 1.0 192 ± 23<br />
54 20.2 ± 2.4 263 ± 32<br />
104 20.4 ± 1.5 7.39 ± 0.46 121.0 ± 3.0 102.8 ± 4.4<br />
150 3.92 ± 0.18 73.0 ± 4.0<br />
201 4.94 ± 0.71 2.99 ± 0.32 21.5 ± 1.9 27.6 ± 3.1<br />
250 1.96 ± 0.13 36.5 ± 1.5<br />
300 1.21 ± 0.18<br />
350 0.88 ± 0.10 3.98 ± 0.79<br />
500 1.83 ± 0.31 0.47 ± 0.12 3.85 ± 0.33 1.83 ± 0.33<br />
slope -2.58 ± 0.12 -3.619 ± 0.044 -4.47 ± 0.14 -5.08 ± 0.29<br />
r 2<br />
0.9553 0.9895 0.9592 0.9901<br />
75
Monovalent cations were reported to significantly increase in vitro translation in<br />
rabbit reticulocyte lysate and K + appeared to have the most suitable radius to support<br />
translation. 228 As the efficiency <strong>of</strong> <strong>eIF4E</strong>-<strong>mRNA</strong> 5'-cap binding decreases at higher K +<br />
concentration, the positive effect caused by K + should rather be attributed to the regulation<br />
<strong>of</strong> other translation components.<br />
Figure 4-17. Determination <strong>of</strong> hydration number ΔN from KCl-dependence <strong>of</strong> the <strong>eIF4E</strong>m<br />
7 GpppG complex formation, at 20°C. <strong>Analysis</strong> according to electrostatic screening theory (solid<br />
line) and stoichiometric cation release <strong>with</strong> the cation number c = 1 (broken line).<br />
An extended analysis, including finite dimensions <strong>of</strong> the interacting molecular<br />
spheres and the hydration effects revealed by the osmotic stress, has been performed for<br />
m 7 GpppG ∗ (Fig. 4-17). The optimal result has been obtained <strong>with</strong>in Davies approximation<br />
for the effective ionic diameter a j = 8 Å, which describes the sum <strong>of</strong> the radii <strong>of</strong> the<br />
interacting species, i.e. three-membered phosphate chain <strong>of</strong> m 7 GpppG and the group <strong>of</strong><br />
four positively charged amino acids localized at the entry <strong>of</strong> the <strong>eIF4E</strong> cap-binding slot<br />
(Arg112, Arg157, Lys162 and Lys206, Fig. 4-13). The product <strong>of</strong> mutually attracting<br />
charges is z = −11.<br />
54 ± 0.<br />
95e.<br />
Previous studies showed that the resultant protein charge<br />
z1 2<br />
log Kas (m 7 GpppG)<br />
8<br />
7<br />
6<br />
5<br />
10 -2 10 -1 10 0<br />
KCl (M)<br />
is only +0.58 e, while a dipole moment is substantial (579 D). 4 Hence, the cap recognizes<br />
directly the binding site <strong>of</strong> <strong>eIF4E</strong> and not the protein as a whole (Fig. 4-18), and the charge<br />
∗ The extended, non-linear analysis required very accurate experimental data for Kas. Those were only<br />
available for m 7 GpppG. The binding constants <strong>of</strong> the remaining cap analogues were determined <strong>with</strong> too<br />
much scattering due to the greater extent <strong>of</strong> wear <strong>of</strong> the xenon impulse lamp <strong>of</strong> the spectr<strong>of</strong>luorometer and<br />
thus insufficient signal-to- noise ratio (< 100:1).<br />
76
localized on the guanine moiety does not by itself play any important role in the first step<br />
<strong>of</strong> the encounter. The negatively charged phosphate chain serves as a molecular anchor,<br />
enabling the cap to form further contacts <strong>with</strong>in the binding site. This conclusion is in a<br />
very good agreement <strong>with</strong> our independent finding that the free energies corresponding to<br />
the phosphate chain interactions are separate from the energy <strong>of</strong> stacking together <strong>with</strong><br />
hydrogen bonds (Table 4-3).<br />
77<br />
Figure 4-18. Molecular electrostatic potential<br />
<strong>of</strong> the <strong>eIF4E</strong> surface at pH 6.0 75 calculated by<br />
<strong>Protein</strong> Explorer 225 in the absence <strong>of</strong> the cap<br />
analogue. The ligand is represented as green<br />
sticks.<br />
4.2.3.1.1.2. Two-Step Mechanism <strong>of</strong> the Cap-<strong>eIF4E</strong> Association<br />
Taken together, the above results provide a molecular interpretation <strong>of</strong> the two-step<br />
mechanism observed for the cap-<strong>eIF4E</strong> association by means <strong>of</strong> stopped-flow<br />
spectroscopy 4 . The first step can be now attributed to the anchoring the phosphate on the<br />
external basic amino acids, and the second step to the interactions deeply inside the m 7 G-<br />
binding slot, i.e. cooperative cation-π stacking and hydrogen bonding <strong>with</strong> Trp56, Trp102<br />
and Glu103. As all these residues are located at the flexible loops, 75 this second step <strong>of</strong> the<br />
complex formation is expected to involve a conformation change.<br />
The two-step model could be verified by mutant analysis, a mirror reflected<br />
methodological approach to the current cap analogue studies. The mutations eliminating all<br />
phosphate interactions should yield the equilibrium Kas for m 7 GTP close to that for m 7 G<br />
(Table 1) and considerably decrease the kinetic encounter rate constant k+1. The complete<br />
cap-slot mutations should retain only the phosphate binding affinity, <strong>with</strong> Kas as for GTP<br />
and the k+1 unchanged. However, some mutations <strong>of</strong> crucial residues can change not only<br />
the nature <strong>of</strong> amino acids, but also the internal structure <strong>of</strong> the protein (local or even global
fold), thus obscuring the investigated effect. By contrast, the studies based on the<br />
chemically modified cap analogues, where each chemical alteration can be individually<br />
controlled, are free from such pitfalls.<br />
4.2.3.1.1.3. Relation to the "Clamping Cycle"<br />
The analysis <strong>of</strong> electrostatic screening confirms a biological role for a putative<br />
phosphate bridge connecting Ser-209 <strong>with</strong> Lys-159. It is thought to "clamp" the previously<br />
bound <strong>mRNA</strong> chain 229 when <strong>eIF4E</strong> is phosphorylated by the eIF4G-associated Mnk1<br />
kinase. 230,231 As the phosphate chain <strong>of</strong> the cap is electrostatically steered toward the<br />
binding site, the mechanism <strong>of</strong> a "clamping cycle", where the phosphorylation happens<br />
after the cap-<strong>eIF4E</strong> binding, 229 is most likely. Otherwise, if the phosphorylation at Ser-209<br />
occurred prior to the cap binding, it would attenuate the electrostatic attraction between<br />
<strong>eIF4E</strong> and the cap. Therefore, the affinity <strong>of</strong> the cap to the previously phosphorylated<br />
protein would be significantly decreased. Recent data showing the effect <strong>of</strong> <strong>eIF4E</strong><br />
phosphorylation on the affinity <strong>of</strong> cap for <strong>eIF4E</strong> 73,74 confirm this conclusion.<br />
4.2.3.1.1.4. Osmotic Stress<br />
Molecular crowding <strong>of</strong> the solution causes a shortage <strong>of</strong> the free water molecules.<br />
The Kas at elevated salt concentration decreases more than it could be explained by<br />
electrostatic screening only (Fig. 4-17), indicating that the binding <strong>of</strong> the cap to <strong>eIF4E</strong><br />
requires additional hydration <strong>of</strong> the molecular surfaces. It should be noted that, although<br />
the activity <strong>of</strong> water at the maximal salt-concentration in the present study was not very far<br />
from unity (aw ~0.97 at 0.5 M KCl), the hydration effects appear quite distinctly, and the<br />
considerable number <strong>of</strong> water molecules that are taken up to the cap-<strong>eIF4E</strong> complex has<br />
been detected: ΔN = 68 ± 16.<br />
The association constant <strong>of</strong> m 7 GpppG extrapolated to the optimal hydration<br />
conditions, i.e. <strong>with</strong>out osmotic stress and electrostatic screening, is<br />
K<br />
as<br />
( 0)<br />
9<br />
−1<br />
= 1.<br />
12 ± 0.<br />
18⋅10<br />
M . It is 150-fold greater than Kas measured in 100 mM KCl,<br />
and is an evidence <strong>of</strong> the strong electrostatic contribution to <strong>eIF4E</strong>-cap binding. On the<br />
other hand, the association constant extrapolated to the pseudostandard state <strong>of</strong> 1M KCl is<br />
K<br />
as<br />
( 1)<br />
4<br />
−1<br />
= 4.<br />
8 ± 1.<br />
9 ⋅10<br />
M . In spite <strong>of</strong> strong electrostatic screening, the intrinsic<br />
thermodynamic preference <strong>of</strong> <strong>eIF4E</strong> for the methylated cap is still efficient. This<br />
78
completely screened interaction <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> m 7 GpppG at 1 M KCl has exactly the same<br />
association constant as m 7 G, the analogue insensitive toward electrostatic interactions,<br />
K<br />
as<br />
7<br />
4<br />
−1<br />
( m G)<br />
= 4.<br />
6 ± 1.<br />
2 ⋅10<br />
M (Table 4-2). Hence, at ionic strength <strong>of</strong> 1 M all the<br />
interactions <strong>with</strong> the phosphates are lost.<br />
4.2.3.1.2. Wyman Linkage <strong>Analysis</strong><br />
In terms <strong>of</strong> Wyman linkage analysis, stoichiometrical ionic interactions are taken<br />
into account in the same manner as excluded volume effects. The most probable process<br />
accompanying the <strong>eIF4E</strong>-cap binding in addition to the change <strong>of</strong> preferential hydration is<br />
potassium cation release 118 from the phosphate chain <strong>of</strong> the cap analogue:<br />
cap( c⋅K + ) + <strong>eIF4E</strong> + ΔN⋅H2O cap<strong>eIF4E</strong>( ΔN⋅H2O) + c⋅K +<br />
4.2.3.1.2.1. Electrolyte Effect<br />
The number <strong>of</strong> K + released from the ligand, c = 0.826 ± 0.092 (Fig. 4-17), indicates<br />
that possibly one K + is displaced from the phosphate chain <strong>of</strong> m 7 GpppG upon binding to<br />
<strong>eIF4E</strong>. Cation release (polyelectrolyte effect) was extensively studied for the entropy-<br />
driven B-DNA interactions <strong>with</strong> oligocations. 118 Large contribution <strong>of</strong> the counterions<br />
displacement into bulk solvent was also reported for anthracycline antibiotic binding to<br />
DNA. 120 Because the cap analogues lacked substantial axial charge distribution, such as<br />
that observed in the helical B-DNA polyanion, the cation dilution effect is much less<br />
pronounced. The contribution <strong>of</strong> the electrolyte effect to the stability <strong>of</strong> the m 7 GpppG-<br />
<strong>eIF4E</strong> complex at 100 mM KCl is ΔG°el = −6.40 ± 0.63 kJ/mol, in comparison <strong>with</strong> the<br />
total free energy <strong>of</strong> m 7 GpppG binding upon these conditions, ΔG° = −38.55 ± 0.15 kJ/mol<br />
(Table 4-2), so the entropic contribution to the association driving force, resulting from the<br />
cation release, is rather moderate (ΔS°el = + 21.8 ± 2.1 J/mol⋅K). The association constant<br />
calculated from the stoichiometrical model and extrapolated to 1M KCl is the same as that<br />
derived from the approach <strong>of</strong> the continuous screening, Kas(1) = 4.45 ± 0.34 ⋅ 10 4 M -1 .<br />
Relatively high Kas ~10 4 <strong>of</strong> m 7 GpppG at 1M KCl and the moderate influence <strong>of</strong> the<br />
electrolyte effect on the binding distinguish the electrostatically attracting cap-<strong>eIF4E</strong><br />
system from the counterion release-driven DNA-oligocation system, for which the<br />
corresponding Kas value was <strong>with</strong>in an order <strong>of</strong> magnitude <strong>with</strong> unity. 118<br />
79
4.2.3.1.2.2. Osmotic Stress<br />
The number <strong>of</strong> water molecules that have to be taken up from the bulk is ΔN = 95 ±<br />
18. Keeping the constant value <strong>of</strong> c = 1 does not cause significant worsening <strong>of</strong> the fit,<br />
according to the Snedecor's F-test (P = 0.11), and gives ΔN = 63.5 ± 9.3. Both <strong>of</strong> these<br />
values agree <strong>with</strong>in the numerical accuracy <strong>with</strong> the hydration effect (ΔN) derived from the<br />
complementary model <strong>of</strong> electrostatic screening, so the two theoretical treatments indicate<br />
the same extent <strong>of</strong> water participation in the cap binding event.<br />
4.2.3.1.3. Comparison <strong>of</strong> the Two Approaches<br />
In respect <strong>of</strong> the direct interaction <strong>with</strong> the electrolyte, the Snedecor's F-test<br />
determines that the screening theory is better (R 2 = 0.9967) than the cation release<br />
approach (R 2 = 0.9906 for c = 1), on the significance level <strong>of</strong> P = 0.0155. Hence, the<br />
description in terms <strong>of</strong> continuous screening by the ionic atmosphere <strong>of</strong> the excess salt is<br />
more relevant. The possible stoichiometric interaction <strong>with</strong> the counterion contributes to<br />
the screening, and additionally provides the advantageous entropic effect to the binding.<br />
4.2.3.1.4. Reasons for KCl-Dependence <strong>of</strong> Internal Rearrangement Rate Constants<br />
Significance <strong>of</strong> the electrostatic forces in kinetics <strong>of</strong> the <strong>eIF4E</strong>-cap complex<br />
formation was also shown by the fluorescence stopped-flow measurements. 4 The encounter<br />
rate constant (k+1) measured experimentally and confirmed by Brownian dynamics<br />
simulations was markedly diminished by the ionic strength (from 4.58 ± 0.18 ⋅10 8 M -1 ⋅s -1 at<br />
50 mM KCl to 0.68 ± 0.04 ⋅10 8 M -1 ⋅s -1 at 350 mM KCl), and the dissociation rate constant<br />
(k-1) was slightly increased (from 30 ± 8 s -1 to 45 ± 7 s -1 , respectively). However, estimates<br />
<strong>of</strong> the second-step rate constants for the internal rearrangement <strong>of</strong> the initial complex (k+2<br />
and k−2) revealed surprisingly analogous ionic strength dependence (from 124 to 73 s -1 , and<br />
from 1 to 12 s -1 , respectively). This intriguing effect could not be explained when<br />
neglecting the excluded volume effects. Now, in light <strong>of</strong> the osmotic stress analysis, the<br />
decrease <strong>of</strong> the second-step rate constant k+2 and the increase <strong>of</strong> k−2 is readily explicable in<br />
terms <strong>of</strong> the conformational change, upon which the significant number <strong>of</strong> water molecules<br />
have to hydrate the protein surface, which becomes more difficult at the elevated osmotic<br />
stress.<br />
80
4.2.3.1.5. Discussion <strong>of</strong> Hydration Effects<br />
For several years increasing attention was paid both to the activity <strong>of</strong> water and to the<br />
ions that can participate in the intermolecular interactions. 117,126,232,233 Additional hydration<br />
can accompany the ligand binding by the proteins that show simultaneous conformational<br />
changes. For instance, the hydration <strong>of</strong> haemoglobin by 60–65 water molecules occurs<br />
<strong>with</strong> the transition from the deoxy T to fully oxygenated R state. 119 Only one chlorine<br />
anion is released upon this transition. Recently, hydration changes that accompany the<br />
binding <strong>of</strong> several intercalators to DNA were exhaustively studied 121 . It was found that<br />
from 0 to 30 water molecules are taken up during complex formation, depending on the<br />
intercalator type. On the other hand, the entropy-driven association <strong>of</strong> lac repressor to lac<br />
operator DNA is accompanied by the release <strong>of</strong> ~200 water molecules. 114 In the cap-<strong>eIF4E</strong><br />
system, the additional hydration similar to that accompanying the haemoglobin<br />
oxygenation is observed. The total number <strong>of</strong> water molecules taken up from the bulk<br />
solvent (~ 65) is much greater than that trapped inside the cap-binding centre, found by<br />
crystallography (9 and 16, in the complex <strong>with</strong> m 7 GDP 75 and m 7 GpppG, 1 respectively, Fig.<br />
4-19), and apparently comparable <strong>with</strong> the amount <strong>of</strong> total water bound per one protein<br />
molecule. 75,91 It should be noted, however, that the water found in the X-ray diffraction<br />
structures is usually an integral part <strong>of</strong> the macromolecular structure and is located in its<br />
internal cavities. The water taken up from the bulk solvent during the complex formation<br />
refers not only to the trapped water but also to the changes <strong>of</strong> the first-layer hydration shell<br />
<strong>of</strong> the molecular surfaces. 234 Such peripheral water is highly unstructured, due mainly to<br />
fluctuations <strong>of</strong> the water molecules imposed by the protein hydrophobic groups.<br />
Figure 4-19. 16 water molecules (magenta spheres, van der Waals radii) were found by<br />
crystallography in the <strong>eIF4E</strong>-m 7 GpppG complex deeply inside the cap-binding pocket. Molecular<br />
solvent accessible surfaces (beige for <strong>eIF4E</strong> and bluish for m 7 GpppG, probe radius <strong>of</strong> 1.4 Å) show<br />
that these water molecules are tightly closed in the protein cavity by the ligand which serves as a<br />
molecular stopper, and thus cannot be regarded as a part <strong>of</strong> the dynamic first-layer hydration shell.<br />
81
4.2.3.1.6. Conformational Change <strong>of</strong> <strong>eIF4E</strong> upon Cap Binding<br />
Such extensive hydration <strong>of</strong> <strong>eIF4E</strong> would respond to exposure <strong>of</strong> additional ~450 Å 2<br />
<strong>of</strong> the molecular surface area to contact <strong>with</strong> water 232 . The exposure <strong>of</strong> the surface requires<br />
a substantial conformational change <strong>of</strong> the protein. This hypothesis is supported by several<br />
independent facts, i.e. by the rearrangement <strong>of</strong> the encounter complex, 4 by the pr<strong>of</strong>ound<br />
(~65%) quenching <strong>of</strong> the intrinsic fluorescence <strong>of</strong> <strong>eIF4E</strong> upon cap binding, by significantly<br />
different fluorescence quenching patterns when comparing interactions <strong>of</strong> apo-<strong>eIF4E</strong> and<br />
cap-saturated <strong>eIF4E</strong> <strong>with</strong> the 4E-BP1 and eIF4G peptides (see 4.3., p. 86), and by<br />
deaggregation <strong>of</strong> <strong>eIF4E</strong> molecules (residues 33-217) forced by the cap analogues binding,<br />
observed by means <strong>of</strong> Dynamic Light Scattering (Fig. 4-20). Conformational changes <strong>of</strong> a<br />
homologous plant eIF(iso)4F complex upon binding <strong>of</strong> m 7 GpppG were also observed by<br />
means <strong>of</strong> the fluorescence stopped-flow measurements. 235<br />
4.2.3.1.6.1. Deaggregation <strong>of</strong> <strong>eIF4E</strong> Induced by Cap Binding<br />
The apo-form <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> an N-terminal tail shorter by 5 amino acids than that<br />
used for the fluorescence studies formed huge aggregates and the solution was totally<br />
polydispersed, as checked by DLS. Only rough estimates <strong>of</strong> the molecular weight (MW)<br />
and the hydrodynamic radius (Rh) <strong>of</strong> the <strong>eIF4E</strong> aggregates could be registered. After<br />
addition <strong>of</strong> one <strong>of</strong> cap analogues (m 7 GTP, p-Cl-bz 7 GTP, m3 227 GTP, m 7 GpppG,<br />
m 7 GppppG, or m 7 Gppppm 7 G) to the <strong>eIF4E</strong> samples progressive deaggregation <strong>with</strong> the<br />
time <strong>of</strong> incubation <strong>with</strong> the cap analogue has been observed. Exemplary results are shown<br />
in Fig. 4-20. The estimates <strong>of</strong> the molecular weight and the hydrodynamic radius <strong>of</strong> the<br />
aggregates dropped after several hours down to the well defined, reliably measured values.<br />
The efficiency <strong>of</strong> the process correlated <strong>with</strong> the affinity <strong>of</strong> the cap analogue for <strong>eIF4E</strong>.<br />
After 24 hours <strong>of</strong> incubation <strong>with</strong> m 7 GTP the protein molecules was sufficiently<br />
monodispersed, characterized by MW = 23.3 kDa and Rh = 2.36 nm, which corresponded<br />
to the monomeric state <strong>of</strong> the protein in the complex <strong>with</strong> m 7 GTP (MWcx ≈ 22.2 kDa,<br />
dimensions: 41 Å × 36 Å × 45 Å, as determined by crystallography 75 ), while <strong>eIF4E</strong> in the<br />
presence <strong>of</strong> m3 227 GTP was polydispersed, and the apo-protein remained still totally<br />
aggregated. Hence, deaggregation <strong>of</strong> <strong>eIF4E</strong> has occurred under the influence <strong>of</strong> interaction<br />
<strong>with</strong> cap.<br />
82
Autocorrelation<br />
coefficient<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
10 -6<br />
10 -5<br />
10 -4<br />
Delay time (μs)<br />
(a) 10 (b)<br />
5<br />
Figure 4-20. Deaggregation <strong>of</strong> <strong>eIF4E</strong> (33-217) at 1 mg/ml induced by the cap analogue binding.<br />
(a) The autocorrelation function becomes well defined <strong>with</strong> the time <strong>of</strong> incubation <strong>with</strong> the 50-fold<br />
excess <strong>of</strong> m 7 GTP ( , apo-protein; , after 1-hour incubation; , after 24-hour incubation). (b) The<br />
estimates <strong>of</strong> the molecular weight (MW, ) and <strong>of</strong> the hydrodynamic radius (Rh, ) <strong>of</strong> non-specific<br />
protein aggregates decrease systematically in the time <strong>of</strong> incubation <strong>with</strong> m 7 GTP by several orders<br />
<strong>of</strong> magnitude to the final values that are characteristic for the single <strong>eIF4E</strong>-cap complex.<br />
4.2.3.2. Ionic Equilibria in <strong>eIF4E</strong>-Cap Complex<br />
7-methylguanosine is a mixture <strong>of</strong> positively charged and zwitterionic forms (due to<br />
dissociation <strong>of</strong> N(1)-H, pKa ~7.24-7.54), the proportions being slightly dependent on the<br />
secondary substituents and the length <strong>of</strong> the phosphate chain. 177 The drop <strong>of</strong> the pKa value<br />
by approximately 2 units, which accompanies the 7-substitution <strong>of</strong> guanosine was<br />
suspected to have a biological significance. The zwitterionic form <strong>of</strong> 7-methylated guanine<br />
was initially postulated to be responsible for interaction <strong>with</strong> <strong>eIF4E</strong>. 156 In contrast,<br />
structural data indicated that the cationic form <strong>of</strong> m 7 GDP binds to <strong>eIF4E</strong>, because <strong>of</strong> the<br />
spatial distances suitable for formation <strong>of</strong> a hydrogen bond between solvent accessible<br />
Glu103 and N(1)-H <strong>of</strong> 7-methylguanosine in the crystal structures (PDB ID codes: 1EJ1,<br />
1AP8) 75,76 . This hydrogen bond is also unambiguously present in the ternary complex <strong>of</strong><br />
<strong>eIF4E</strong> bound to m 7 GDP and eIF4GII peptide, although the crystals were obtained at pH 8.5<br />
(PDB ID code: 1EJH) 91 . However, the N(1)-H proton was not directly observed during the<br />
NMR experiment, 76 so the issue was still not entirely resolved.<br />
10 -3<br />
10 -2<br />
The affinity <strong>of</strong> <strong>eIF4E</strong> for m 7 GTP as a function <strong>of</strong> pH (Table 4-5, Fig. 4-21) shows<br />
the optimal binding at pH 7 . 24 ± 0.<br />
13,<br />
close to the physiological pH <strong>of</strong> 7.4, and lower than<br />
the pH 7.6 reported for the full length <strong>eIF4E</strong> from human erythrocytes, purified by m 7 G-<br />
affinity chromatography. 156 The observed pH-dependence <strong>of</strong> Kas is rather flat, providing a<br />
wide pH-range <strong>of</strong> efficient cap-<strong>eIF4E</strong> binding. From the Wyman model, 193 comprising one<br />
site to be protonated and one site to be deprotonated, two effective acidic dissociation<br />
83<br />
Rh (nm), MW (kDa)<br />
10 6<br />
10 4<br />
10 3<br />
10 2<br />
10 1<br />
10 0<br />
MW<br />
R h<br />
0 6 12 18 24<br />
Time <strong>of</strong> incubation <strong>with</strong> m 7 GTP (h)<br />
23.3<br />
2.36
constants have been determined: = 7.<br />
99 ± 0.<br />
14 for N(1)-H <strong>of</strong> the ligand base moiety<br />
pK L<br />
in the presence <strong>of</strong> the protein, and = 6.<br />
49 ± 0.<br />
13 for the hydroxyl group <strong>of</strong> Glu103 in<br />
pK P<br />
the binding site <strong>of</strong> <strong>eIF4E</strong>, in the presence <strong>of</strong> the ligand (the errors <strong>of</strong> pKs are numerical).<br />
Taking into account experimental uncertainties as well as simplifications <strong>of</strong> the model, the<br />
real accuracy <strong>of</strong> pKs is close to 1 pH unit. Making the number <strong>of</strong> protonated and<br />
deprotonated sites as free parameters <strong>of</strong> the fitted function leads to negligible improvement<br />
<strong>of</strong> the results, on the basis <strong>of</strong> the statistical Snedecor's F-test. The elevated effective pKL in<br />
relation to the pKL value in the absence <strong>of</strong> the interacting counterpart shows that m 7 G<br />
exists in a cationic form in the <strong>eIF4E</strong>-bound state and donates N(1)-H proton for the<br />
hydrogen bond <strong>with</strong> the deprotonated carboxyl group <strong>of</strong> Glu103. Such an increase <strong>of</strong> pKL<br />
caused by the chemical microenvironment <strong>of</strong> the negatively charged Glu103 was<br />
postulated on the basis <strong>of</strong> the crystallographic structure. 75<br />
Table 4-5. Association constants for m 7 GTP<br />
binding to <strong>eIF4E</strong> as a function <strong>of</strong> pH, at<br />
20 °C, 100 mM KCl.<br />
pH Kas ⋅ 10 -6 (M -1 )<br />
6.32 53.7 ± 4.4<br />
6.56 74.4 ± 7.7<br />
6.78 96.9 ± 8.4<br />
7.01 97.9 ± 8.4<br />
7.20 108.7 ± 9.2<br />
7.42 102.3 ± 6.5<br />
7.61 82.1 ± 11.5<br />
7.84 74.2 ± 4.3<br />
8.02 74.7 ± 6.7<br />
log Kas (m 7 GTP)<br />
84<br />
Figure 4-21. Binding affinity <strong>of</strong> m 7 GTP to<br />
<strong>eIF4E</strong> as a function <strong>of</strong> pH, at 20°C.<br />
6.0 6.5 7.0 7.5 8.0 8.5<br />
The free energy <strong>of</strong> binding between the species in their appropriate ionic states (pH-<br />
independent ΔG°) is: ΔG° pH-ind = −45.765 ± 0.017 kJ/mol, and the energetic costs <strong>of</strong><br />
keeping the cationic state <strong>of</strong> m 7 GTP and the anionic state <strong>of</strong> Glu103 in the <strong>eIF4E</strong> binding<br />
site are very small: ΔG°L = +0.368 ± 0.11 kJ/mol and ΔG°P = +0.435 ± 0.12 kJ/mol,<br />
respectively. Even after the increase <strong>of</strong> the pKP <strong>of</strong> Glu103 to ~ 6 units inside the protein,<br />
pKL and pKP are separated enough to allow tight m 7 GTP-<strong>eIF4E</strong> binding at pH 7.2,<br />
characterized by ΔG° = −45.099 ± 0.088 kJ/mol (Table 4-2) very close to ΔG° pH-ind .<br />
8.5<br />
8.0<br />
7.5<br />
7.0<br />
pH
In summary, the decrease <strong>of</strong> pKa <strong>of</strong> guanosine as a result <strong>of</strong> methylation at N(7) is a<br />
side-effect, which must be cancelled by the microenvironment <strong>of</strong> the protein binding site.<br />
Instead, the biological importance <strong>of</strong> the 7-methylation consists in strong enhancement <strong>of</strong><br />
stacking between the cationic m 7 G and the aromatic side chains (cation - π stacking),<br />
which stabilizes the base moiety enough to allow formation <strong>of</strong> the three hydrogen bonds in<br />
the binding site (see 4.2.2., p. 64).<br />
4.2.4. Conclusions<br />
The results reveal individual role <strong>of</strong> various structural elements <strong>of</strong> the cap in the two-<br />
step process <strong>of</strong> <strong>eIF4E</strong>-cap recognition. The 5'-phosphate chain is the primary anchor to<br />
<strong>eIF4E</strong>. The <strong>eIF4E</strong>-cap binding is accomplished by the specific contacts <strong>of</strong> three phosphates<br />
and m 7 G moiety but not <strong>of</strong> the second guanosine. The biological importance <strong>of</strong><br />
methylation <strong>of</strong> the first guanosine at N(7) has been elucidated by showing that the<br />
thermodynamic origin <strong>of</strong> the strong discrimination between 7-methylated and<br />
unmethylated counterparts (5000-fold difference in Ka at 20°C) consists in absolute<br />
cooperativity <strong>of</strong> cation-π stacking and hydrogen bonding during the second step <strong>of</strong> the<br />
complex formation. The enhanced stacking between the cationic m 7 G and the aromatic Trp<br />
side chains is necessary to stabilize effectively the base moiety, which is a precondition for<br />
formation <strong>of</strong> three hydrogen bonds inside the cap-binding slot. <strong>eIF4E</strong> selects precisely<br />
between the 7-monomethylguanine and 2,2,7-trimethylguanine analogues (760-fold<br />
difference in Kas). Possibilities <strong>of</strong> cation release and water exchange that could accompany<br />
the binding have been also checked. The estimated water uptake <strong>of</strong> ~65 water molecules to<br />
the cap-binding slot and to the hydration shell <strong>of</strong> the complex is relevant to conformational<br />
rearrangement <strong>of</strong> entire <strong>eIF4E</strong> upon the second step <strong>of</strong> cap binding.<br />
85
4.3. Molecular <strong>Interaction</strong>s <strong>with</strong>in Ternary Complexes<br />
Involving <strong>eIF4E</strong>, a Cap Analogue, and an <strong>eIF4E</strong>-Binding Motif<br />
from eIF4G or 4E-BP1 <strong>Protein</strong>s<br />
The fluorescence affinity measurements have been extended to include ternary<br />
complexes which consisted <strong>of</strong> <strong>eIF4E</strong>, a cap-analogue, and a synthetic peptide<br />
encompassing the <strong>eIF4E</strong> recognition motif from the mammalian proteins eIF4GI, eIF4GII<br />
and 4E-BP1. The 4E-BP1 peptides were either unphosphorylated or monophosphorylated<br />
at Ser65 and diphosphorylated at Ser65/Thr70. The eIF4G and 4E-BP1 proteins bind to the<br />
convex dorsal surface <strong>of</strong> <strong>eIF4E</strong>, on the opposite side <strong>of</strong> the protein in respect to the cap-<br />
binding slot (Fig. 4-22). Application <strong>of</strong> the precise synchronized titration method allowed<br />
to test the putative cooperation between the two distant binding sites <strong>of</strong> <strong>eIF4E</strong>, 166,167 and<br />
the influence <strong>of</strong> phosphorylation at Ser65 and Thr70 on regulation <strong>of</strong> 4E-BP1 binding to<br />
<strong>eIF4E</strong>.<br />
(a) (b)<br />
Figure 4-22. The dorsal surface <strong>of</strong> <strong>eIF4E</strong> coloured according to the molecular electrostatic<br />
potential 225 in the absence <strong>of</strong> ligands (red – negative, blue – positive). The attached peptide is<br />
shown in the ribbon representation and corresponds to the <strong>eIF4E</strong> recognition sequence <strong>of</strong> (a) 4E-<br />
BP1 (yellow, at pH 7.5) or (b) eIF4GII (orange, at pH 8.5). 91 The peptide tyrosine is shown as balls<br />
and sticks. Trp73 <strong>of</strong> <strong>eIF4E</strong> is shown in the van der Waals spacefill representation and coloured<br />
green. Orientation <strong>of</strong> the two aromatic residues is almost ideally perpendicular. A fragment <strong>of</strong><br />
bound m 7 GDP (oxygens <strong>of</strong> the β phosphate group, red spheres) is visible in the bottom right part <strong>of</strong><br />
each figure.<br />
86
4.3.1. <strong>eIF4E</strong> Fluorescence Quenching Pattern upon Peptide Binding<br />
Association <strong>of</strong> the eIF4GI, eIF4GII or 4E-BP1 peptides <strong>with</strong> <strong>eIF4E</strong> leads to<br />
quenching <strong>of</strong> the <strong>eIF4E</strong> intrinsic fluorescence, mostly at longer wavelengths (355 – 360<br />
nm) than quenching upon association <strong>with</strong> cap (330 – 340 nm). This is caused primarily by<br />
interaction <strong>of</strong> the peptides <strong>with</strong> the water accessible Trp73 <strong>of</strong> <strong>eIF4E</strong>. This amino acid is<br />
thought to be the most important residue <strong>of</strong> the phylogenetically invariant dorsal surface<br />
region, since it interacts <strong>with</strong> three peptide amino acid side chains: Leu(5), Leu(6) and<br />
Gln(9) <strong>of</strong> eIF4GII, or Leu(5), Met(6) and Arg(9) <strong>of</strong> 4E-BP1 91 (Fig. 4-23, numbering in<br />
relation to the invariant Tyr). Mutation <strong>of</strong> Trp73 to Ala was found to prevent productive<br />
interactions <strong>with</strong> 4E-BP1 and eIF4GI in a biological assay. 230<br />
(a)<br />
(b)<br />
Figure 4-23. interatomic contacts between Trp73 <strong>of</strong> <strong>eIF4E</strong> (green sticks) and the peptides in the<br />
crystal structures. 91 The peptide amino acid side chains that interact <strong>with</strong> Trp73 are shown as thick<br />
sticks in the CPK colours. (a) Leu(5), Leu(6) and Gln(9) <strong>of</strong> the eIF4GII peptide; (b) Leu(5), Met(6)<br />
and Arg(9) <strong>of</strong> 4E-BP1. Remaining parts <strong>of</strong> the peptides are represented by the backbone. The<br />
hydrogen bond and the van der Waals bonds <strong>with</strong> their lengths are drawn as dotted lines.<br />
87
To analyse the <strong>eIF4E</strong> binding affinity <strong>of</strong> the peptides and check whether cap-binding<br />
and 4E-BP/eIF4G-binding centres are cooperative, cross-titration experiments <strong>with</strong> each<br />
peptide have been performed (Fig. 4-24). Comparison <strong>of</strong> the emission band changes for<br />
saturation <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> a cap analogue or <strong>with</strong> an oligopeptide, and <strong>with</strong> both <strong>of</strong> them is<br />
shown in Fig. 4-25. Titration curves for formation <strong>of</strong> <strong>eIF4E</strong> binary and ternary complexes<br />
<strong>with</strong> m 7 GTP and the oligopeptides are presented in Fig. 4-26, 27, 28. Irrespective <strong>of</strong> the<br />
succession <strong>of</strong> the cap and the peptide added, the total final quenching after saturation <strong>of</strong><br />
both <strong>eIF4E</strong> binding sites is always identical. However, the partial quenching (Q) <strong>of</strong> the<br />
total initial fluorescence signal, which corresponds to saturation <strong>of</strong> one <strong>of</strong> the <strong>eIF4E</strong><br />
binding sites, <strong>with</strong> and <strong>with</strong>out the prior saturation <strong>of</strong> the other, is different (Fig. 4-25,<br />
Table 4-6). The fluorescence quenching upon titration <strong>with</strong> m 7 GTP <strong>of</strong> the peptide-saturated<br />
<strong>eIF4E</strong> is < 60 %, while the apo-protein reveals ~ 65 % <strong>of</strong> quenching. The quenching<br />
resulting from the peptides binding is much smaller, ~ 12 % for m 7 GTP-saturated <strong>eIF4E</strong><br />
and ~ 19 % for the apo-protein. The differences between Q(apo) and Q(sat) are ~ 3 % for<br />
the unphosphorylated 4E-BP1 peptide, ~ 9 % for the phosphorylated 4E-BP1 peptides and<br />
~ 7 % for eIF4G peptides. This succession- and structure-dependent behaviour <strong>of</strong> the<br />
quenching patterns supports previous conclusions pointing to the conformational change <strong>of</strong><br />
<strong>eIF4E</strong> upon cap binding, and a possible conformational change upon the peptide binding.<br />
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 10 20 30 40 50 60 70<br />
Time (min)<br />
<strong>eIF4E</strong> + 4GI + m 7 GTP<br />
<strong>eIF4E</strong> + m 7 GTP + 4GI<br />
Figure 4-24. A typical time course <strong>of</strong> the fluorescence intensity for the cross-titration experiment<br />
<strong>of</strong> the ternary complex formation, at 20°C.<br />
Data points ( ): 0-5 min, initial baseline; 6-38 min, titration <strong>with</strong> eIF4GI peptide; 39 min,<br />
intermediate baseline; 40-71 min, titration <strong>with</strong> m 7 GTP; 72-76 min, final baseline.<br />
Data points ( ): 0-5 min, initial baseline; 6-37 min, titration <strong>with</strong> m 7 GTP; 38 min, intermediate<br />
baseline; 39-71 min, titration <strong>with</strong> eIF4GI peptide; 72-76 min, final baseline.<br />
88
Fluorescence (a. u.)<br />
ΔΔ F (%)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
300 320 340 360 380 400<br />
20<br />
0<br />
-20<br />
-40<br />
-60<br />
Wavelength (nm)<br />
Figure 4-25. (a) Fluorescence emission spectra excited at 290 nm <strong>of</strong> apo-<strong>eIF4E</strong> (——), <strong>eIF4E</strong> in<br />
the binary complex <strong>with</strong> the 4E-BP1 P-Ser65 peptide (-----), <strong>eIF4E</strong> in the binary complex <strong>with</strong><br />
m 7 GTP (− − −), <strong>eIF4E</strong> in the ternary complex <strong>with</strong> m 7 GTP and the peptide (− - −), and <strong>of</strong> the pure<br />
peptide (). Application <strong>of</strong> the emission wavelength <strong>of</strong> 355 nm allows for elimination <strong>of</strong> the<br />
direct fluorescence signal originating from peptide tyrosine during titration. (b) Differential spectra<br />
corresponding to saturation <strong>of</strong> the <strong>eIF4E</strong>-m 7 GTP complex <strong>with</strong> the 4E-BP1 P-Ser65 peptide (− - −),<br />
saturation <strong>of</strong> apo-<strong>eIF4E</strong> <strong>with</strong> the peptide (-----), saturation <strong>of</strong> the <strong>eIF4E</strong>-peptide complex <strong>with</strong><br />
m 7 GTP (− − −), saturation <strong>of</strong> apo-<strong>eIF4E</strong> <strong>with</strong> m 7 GTP(− - - −), and saturation <strong>of</strong> apo-<strong>eIF4E</strong> <strong>with</strong><br />
both m 7 GTP and the peptide (——).<br />
89<br />
(a)<br />
300 320 340 360 380 400<br />
Wavelength (nm)<br />
(b)
Table 4-6. Equilibrium association constants (Kas) and partial quenching (Q) <strong>of</strong> total initial<br />
fluorescence upon formation <strong>of</strong> the binary complexes <strong>of</strong> the apo-form <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the eIF4GI,<br />
eIF4GII and 4E-BP1 peptides, and <strong>of</strong> the ternary complexes (m 7 GTP-<strong>eIF4E</strong>-peptide) <strong>of</strong> previously<br />
m 7 GTP- or peptide-saturated <strong>eIF4E</strong>, at 20°C. For reference the association constant <strong>of</strong> m 7 GTP to<br />
apo-<strong>eIF4E</strong> Kas = 108.7 ± 4.0 ⋅ 10 6 M -1 (Table 4-2), quenching Q ~ 65 %.<br />
Peptides: eIF4GI eIF4GII<br />
Kas ⋅ 10 -6 (M -1 ) for the peptides<br />
<strong>eIF4E</strong>(m 7 GTP-saturated) 21.43 ± 2.15 5.35 ± 0.20<br />
<strong>eIF4E</strong>(apo) 13.65 ± 0.86 6.51 ± 0.19<br />
Kas ⋅ 10 -6 (M -1 ) for m 7 GTP<br />
<strong>eIF4E</strong>(peptide-saturated) 120.5 ± 7.2 137.9 ± 15.8<br />
Fluorescence quenching Q (% <strong>of</strong> total initial fluorescence)<br />
<strong>eIF4E</strong>(m 7 GTP-saturated) 12.1 ± 0.9 13.6 ± 2.1<br />
<strong>eIF4E</strong>(apo) 18.3 ± 1.2 20.6 ± 2.5<br />
Peptides: 4E-BP1 4E-BP1 4E-BP1<br />
(P-Ser65) (P-Ser65/Thr70)<br />
Kas ⋅ 10 -6 (M -1 ) for the peptides<br />
<strong>eIF4E</strong>(m 7 GTP-saturated) 9.47 ± 0.39 4.77 ± 0.40 5.72 ± 0.35<br />
<strong>eIF4E</strong>(apo) 10.59 ± 0.84 5.91 ± 0.44 5.74 ± 0.34<br />
Kas ⋅ 10 -6 (M -1 ) for m 7 GTP<br />
<strong>eIF4E</strong>(peptide-saturated) 101.6 ± 7.3 107.4 ± 5.2 101.0 ± 8.6<br />
Fluorescence quenching Q (% <strong>of</strong> total initial fluorescence)<br />
<strong>eIF4E</strong>(m 7 GTP-saturated) 15.1 ± 3.6 10.2 ± 5.5 11.3 ± 4.6<br />
<strong>eIF4E</strong>(apo) 18.2 ± 9.5 18.8 ± 9.0 20.2 ± 1.4<br />
4.3.2. Cooperativity in Ternary Peptide-<strong>eIF4E</strong>-Cap Complexes<br />
In general, the image <strong>of</strong> interactions <strong>with</strong>in ternary complexes is unsymmetrical,<br />
since binding <strong>of</strong> one ligand (e. g. the peptide) can modify the protein affinity for the other<br />
(the cap analogue), but this is not necessarily accompanied by the evident influence in the<br />
reverse direction. Hence, there is no simple thermodynamic cycle that would describe<br />
binding <strong>of</strong> the two ligands to <strong>eIF4E</strong>.<br />
90
4.3.2.1. Cooperativity <strong>of</strong> Cap and eIF4G Peptides Binding<br />
The fluorometrically determined values <strong>of</strong> the association constants have been used<br />
to address the cooperativity problem by a direct quantitative comparison. All the <strong>eIF4E</strong>-<br />
peptide Kas are in the order <strong>of</strong> 10 7 M -1 (Table 4-6). The Kas values for binding <strong>of</strong> cap-<br />
saturated <strong>eIF4E</strong> to eIF4GI and eIF4GII peptides (21.43 μM -1 and 5.35 μM -1 , respectively)<br />
are close to those derived by Isothermal Titration Calorimetry 91 (ITC) (37 μM -1 and 6.7<br />
μM -1 , respectively). The peptide which binds the tightest to <strong>eIF4E</strong> is eIF4GI and its<br />
interaction is few but unambiguously influenced by the apo- or cap-saturated state <strong>of</strong><br />
<strong>eIF4E</strong>, i.e. an 1.6-fold increase <strong>of</strong> Kas has been observed for the eIF4GI peptide binding to<br />
cap-saturated <strong>eIF4E</strong>. A similar effect, albeit to a lesser extent, appears also for the eIF4GII<br />
peptide. Reversely, a slight enhancement <strong>of</strong> the m 7 GTP-affinity (only up to 1.3-fold,<br />
quantitatively beyond one standard deviation but <strong>with</strong>in two standard deviations) has been<br />
noted after previous saturation <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> eIF4GI or eIF4GII peptides.<br />
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
Figure 4-26. Titration <strong>with</strong> m 7 GTP <strong>of</strong> apo-<strong>eIF4E</strong> () and <strong>of</strong> the <strong>eIF4E</strong>-eIF4GI peptide complex<br />
(); 20 °C, excitation wavelength 290 nm, emission wavelength 355 nm.<br />
The case for the cooperativity is based on several experimental results:<br />
1. The eIF4GI protein was found to strongly enhance the crosslinking effectivity <strong>of</strong> <strong>eIF4E</strong><br />
to the <strong>mRNA</strong> 5'-cap structure. 216<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
m 7 GTP (μM)<br />
2. Mutation <strong>of</strong> Trp166 to Ala in the cap-binding slot resulted in the protein which was<br />
unable to bind either eIF4G or 4E-BPs at its dorsal surface. Hence, it can be concluded<br />
91<br />
<strong>eIF4E</strong>+eIF4GI<br />
<strong>eIF4E</strong>
Fluorescence (a. u.)<br />
Fluorescence (a. u.)<br />
100<br />
90<br />
80<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
eIF4GI peptide ( μM)<br />
100<br />
90<br />
80<br />
(a)<br />
(b)<br />
Figure 4-27. (a) Titration <strong>with</strong> the eIF4GI peptide <strong>of</strong> <strong>eIF4E</strong> ( ) and <strong>of</strong> the <strong>eIF4E</strong>-m 7 GTP complex<br />
( ). (b) Titration <strong>with</strong> the eIF4GII peptide <strong>of</strong> <strong>eIF4E</strong> ( ) and <strong>of</strong> the <strong>eIF4E</strong>-m 7 GTP complex ( ). 20<br />
°C, excitation wavelength 290 nm, emission wavelength 355 nm.<br />
that this cap-binding residue plays a role in maintaining the internal structure <strong>of</strong> <strong>eIF4E</strong><br />
necessary for recognition <strong>of</strong> the eIF4G proteins and <strong>of</strong> 4E-BPs. 236<br />
3. Among nine crystallographically independent structures <strong>of</strong> the m 7 GDP-<strong>eIF4E</strong> binary 75<br />
and ternary 91 complexes, and the m 7 GpppG complex, 1 there are two cases where the C-<br />
terminal loop (206-213) located in the vicinity <strong>of</strong> the cap-binding site has fully ordered<br />
backbone, and the N-terminus (28-35) close to the eIF4G/4E-BP binding site is<br />
concurrently ordered. In the remaining cases, both the loop and the N-tail are<br />
92<br />
<strong>eIF4E</strong>+m<br />
<strong>eIF4E</strong><br />
7 GTP<br />
<strong>eIF4E</strong>+m<br />
<strong>eIF4E</strong><br />
7 GTP<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
eIF4GII peptide ( μM)
unordered. These could indirectly suggest a possibility <strong>of</strong> cooperation between the two<br />
distant binding sites.<br />
On the other hand:<br />
1. Comparison <strong>of</strong> the crystallographic structures <strong>of</strong> binary and ternary complexes revealed<br />
no important structural differences <strong>with</strong>in the <strong>eIF4E</strong>-cap binding slot in the absence or<br />
presence <strong>of</strong> either the eIF4GII or 4E-BP1 peptide. 75,91<br />
2. Preliminary fluorescence study suggested no significant changes <strong>of</strong> the association<br />
constant for binding <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the cap analogue in the presence <strong>of</strong> a 17-aminoacid<br />
mammalian eIF4GII peptide. 237<br />
Solution <strong>of</strong> the apparent discrepancies came from two sets <strong>of</strong> experiments.<br />
1. Recently, it was shown by the gel-shift experiments and m 7 GTP-Sepharose binding<br />
that a 17-mer fragment <strong>of</strong> yeast eIF4GI exerted a negligible influence on the <strong>eIF4E</strong>-cap<br />
interaction, while complete eIF4GI and its larger fragments produced distinct<br />
enhancement <strong>of</strong> the <strong>eIF4E</strong>-cap binding. 168<br />
2. Finally, the quantitative results reported herein confirm that the putative positive<br />
cooperativity is possible, and that the minimal <strong>eIF4E</strong>-binding motifs <strong>of</strong> the eIF4G<br />
proteins can be alone insufficient to induce such spatial changes which could be<br />
evident in the crystal structures <strong>of</strong> the cap-binding site. 91<br />
4.3.2.2. Cooperativity <strong>of</strong> Cap and 4E-BP1 Peptides Binding<br />
In case <strong>of</strong> the 4E-BP1 peptides, the affinity for the apo-protein is the same as that for<br />
the cap-saturated <strong>eIF4E</strong> (Table 4-6, p. 90). The Kas values for the unphosphorylated 17-mer<br />
peptide 4E-BP1 (9.47 ⋅ 10 6 M -1 and 10.59 ⋅ 10 6 M -1 ) agree well <strong>with</strong> Kas calculated from<br />
ITC 91 (20 ⋅ 10 6 M -1 ). ITC for the full length 4E-BP1 yielded Kas 7-fold higher (67 ⋅ 10 6 M -<br />
1 ). In terms <strong>of</strong> the binding free energy, however, this difference between ΔG° for the full<br />
length protein (−43.93 kJ/mol) and the peptide (−39.16 kJ/mol) is small, what indicates<br />
that majority <strong>of</strong> the intermolecular contacts required for stabilization <strong>of</strong> the 4E-BP1-<strong>eIF4E</strong><br />
complex are accomplished in the model system <strong>with</strong> participation <strong>of</strong> the short peptide.<br />
Binding <strong>of</strong> full length 4E-BP1 and 4E-BP2 to <strong>eIF4E</strong> was reported to enhance the<br />
cap-<strong>eIF4E</strong> association, as suggested from m 7 GTP-affinity chromatography and Surface<br />
Plasmon Resonance 166 (SPR). The reversed cooperativity, i.e. an increase <strong>of</strong> the 4E-BP2<br />
affinity for the cap-saturated <strong>eIF4E</strong> in comparison <strong>with</strong> apo-<strong>eIF4E</strong> was also found using<br />
SPR. 238 The differences in the fluorescence quenching patterns upon formation <strong>of</strong> the<br />
93
ternary complexes (Fig. 4-25, 26, 27, 28, Table 4-6), and arguments discussed above for<br />
the eIF4G peptides points to a putative cooperation between the two binding sites.<br />
However, the binary 4E-BP1 peptide-<strong>eIF4E</strong> and cap-<strong>eIF4E</strong> interactions do not affect the<br />
association constant values for the further formation <strong>of</strong> the ternary peptide-<strong>eIF4E</strong>-cap<br />
complexes, <strong>with</strong>in experimental errors. No measurable affinity changes for the 17-mer and<br />
25-mer fragments <strong>of</strong> 4E-BP1 could reflect the inability <strong>of</strong> the peptides to completely<br />
mimic the influence <strong>of</strong> full length 4E-BP1 on the conformation <strong>of</strong> the <strong>eIF4E</strong> protein. It is<br />
worth noting here that free 4E-BP1 is unfolded in solution unless gets bound to <strong>eIF4E</strong>. 239<br />
4.3.3. Regulation <strong>of</strong> 4E-BP1 Activity by Phosphorylation<br />
Phosphorylation <strong>of</strong> specific serine and threonine residues <strong>of</strong> 4E-binding proteins<br />
modulates their affinity for <strong>eIF4E</strong>. 229 A two-step mechanism <strong>of</strong> 4E-BP1 phosphorylation<br />
was proposed, involving phosphorylation on Thr37 and Thr46 as a priming event for<br />
subsequent phosphorylation <strong>of</strong> Ser65 and Thr70. 92 Recently, it was shown by<br />
phosphopeptide mapping that both <strong>of</strong> these phosphorylation events were insufficient to<br />
disrupt the 4E-BP1 binding <strong>with</strong> <strong>eIF4E</strong>, 3 and the possible sources <strong>of</strong> disagreement <strong>with</strong> the<br />
previous publications were thoroughly discussed. 93-95<br />
The fluorescence quenching experiments for cap-free and cap-saturated <strong>eIF4E</strong><br />
confirm these results (Table 4-6). The monophosphorylation <strong>of</strong> the 4E-BP1 peptide at<br />
Ser65 causes only ~ 2-fold decrease in the affinity for <strong>eIF4E</strong> (ΔΔG° less than +1.7 kJ/mol),<br />
and diphosphorylation at both Ser65/Thr70 does not reduce the affinity further. Mono- and<br />
diphosphorylated 4E-BP1 peptides still retain the high ability to interact <strong>with</strong> <strong>eIF4E</strong>, <strong>with</strong><br />
Kas about 5 ⋅ 10 6 M -1 and ΔG° about −38 kJ/mol. Thus, phosphorylation <strong>of</strong> both Ser65 and<br />
Thr70 is insufficient to abolish the <strong>eIF4E</strong> binding.<br />
However, several authors reported substantial reduction <strong>of</strong> the <strong>eIF4E</strong> association<br />
<strong>with</strong> 4E-BP1 phosphorylated at Ser65 in vitro. 93-95 A nearly two orders <strong>of</strong> magnitude<br />
decrease <strong>of</strong> Kas was observed by means <strong>of</strong> SPR due to phosphorylation at Ser65 <strong>of</strong> the full<br />
length 4E-BP1 mutant in which alanine had been substituted at four out <strong>of</strong> the five<br />
Thr/Ser-P sites, leaving Ser65 susceptible to phosphorylation. 94 A value <strong>of</strong> Kas for the<br />
unphosphorylated mutant was determined as 278 ⋅ 10 6 M -1 . ITC yielded a lower Kas value<br />
<strong>of</strong> 67 ⋅ 10 6 M -1 for the full length 4E-BP1, 91 but at this level <strong>of</strong> the equilibrium constants<br />
94
Fluorescence (a. u.)<br />
Fluorescence (a. u.)<br />
Fluorescence (a. u.)<br />
100<br />
90<br />
80<br />
100<br />
(a)<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
90<br />
80<br />
100<br />
(b)<br />
4E-BP1 peptide ( μM)<br />
Figure 4-28. () Titration <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> (a) the 4E-BP1 peptide, (b) the 4E-BP1 P-Ser65 peptide,<br />
(c) the 4E-BP1 P-Ser65/Thr70 peptide. () Titration <strong>of</strong> the <strong>eIF4E</strong>-m 7 GTP complex <strong>with</strong> the<br />
peptides, as indicated above. 20 °C, excitation wavelength 290 nm, emission wavelength 355 nm.<br />
95<br />
<strong>eIF4E</strong>+m<br />
<strong>eIF4E</strong><br />
7 GTP<br />
<strong>eIF4E</strong>+m<br />
<strong>eIF4E</strong><br />
7 GTP<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
90<br />
80<br />
4E-BP1 P-Ser65 peptide ( μM)<br />
(c)<br />
<strong>eIF4E</strong>+m 7 GTP<br />
<strong>eIF4E</strong><br />
10 -3 10 -2 10 -1 10 0 10 1<br />
4E-BP1 P-Ser65/Thr70 peptide ( μM)
(corresponding to ΔG° about –46 kJ/mol) the 4-fold difference between the Kas values<br />
determined by two different methods is not very significant (ΔΔG° about 3.7 kJ/mol). Even<br />
taking into account the 75-fold drop <strong>of</strong> the association constant to 3.7 ⋅ 10 6 M -1 upon<br />
phosphorylation at Ser65 according to SPR results, 94 the corresponding energy difference<br />
ΔΔG° would be about +10.5 kJ/mol, which is not much in comparison <strong>with</strong> the remaining<br />
binding free energy <strong>of</strong> the monophosphorylated protein (ΔG° about –36.8 kJ/mol).<br />
Discrepancies could partially arise from some experimental problems <strong>with</strong><br />
unphosphorylated 4E-BP1 protein, which lacks folded structure in solution, 239 so the<br />
cysteine residues are not protected against the oxidation. Many times the presence <strong>of</strong> the<br />
4E-BP1 peptide dimers in the stock solution (see 3.2., p. 24) resulted in apparently much<br />
higher values <strong>of</strong> the association constants for the 4E-BP1 peptide than reported in Table 4-<br />
6 (up to 2 orders <strong>of</strong> magnitude, data not shown).<br />
The peptides seem not to be the perfect mimetic models for all properties <strong>of</strong> the full<br />
length protein. However, the previous studies by phosphopeptide mapping demonstrated<br />
that full-length 4E-BP1 monophosphorylated on Ser65 could bind to <strong>eIF4E</strong> efficiently,<br />
while no binding could be detected for 4E-BP1 deleted in the <strong>eIF4E</strong>-binding site. 3 Hence,<br />
the peptides are good models for investigations how single phosphorylation modulates the<br />
intermolecular interactions.<br />
4.3.4. Concluding Remarks<br />
Experiments based on the fluorescence titration studies reconcile the biochemical,<br />
sometimes contradictory, observations that were done hitherto. The results suggest that<br />
cooperation between cap- and eIF4G/4E-BP-binding sites <strong>of</strong> <strong>eIF4E</strong> is possible but hardly<br />
detectable for the short peptides. The <strong>mRNA</strong> cap can be caught more tightly by <strong>eIF4E</strong> in<br />
complex <strong>with</strong> the full length eIF4Gs, and eIF4GI can bind more efficiently to <strong>eIF4E</strong> in<br />
complex <strong>with</strong> RNA.<br />
4E-BP1 monophosphorylated at Ser-65 still possesses the high, submicromolar<br />
affinity for <strong>eIF4E</strong>. Diphosphorylation at Ser-65 and Thr-70 <strong>of</strong> 4E-BP1 has been also<br />
proved insufficient to abrogate 4E-BP1 binding <strong>with</strong> both apo-<strong>eIF4E</strong> and cap-saturated<br />
<strong>eIF4E</strong>. Thus, phosphorylation <strong>of</strong> additional residues, most likely the priming sites Thr37<br />
and Thr46, 92 is required to release 4E-BP1 from <strong>eIF4E</strong>.<br />
96
4.4. <strong>Thermodynamic</strong>s <strong>of</strong> <strong>mRNA</strong> 5' Cap Binding by <strong>eIF4E</strong><br />
Hydrophobic interactions are widely believed to make important contributions to the<br />
stability <strong>of</strong> protein-ligand complexes. A lot <strong>of</strong> studies were devoted to evaluation <strong>of</strong> their<br />
structural and thermodynamic basis for the purpose <strong>of</strong> drug design. Considering that salt<br />
bridges, cation-π stacking, and hydrogen bonds play a dominant role in <strong>mRNA</strong> 5' cap −<br />
<strong>eIF4E</strong> binding 75 , this molecular system is significantly distinct from the hydrophobic ones<br />
which are usually a focus <strong>of</strong> the thermodynamic studies. The cap − <strong>eIF4E</strong> binding is also<br />
accompanied by other processes, i.e. the conformational change <strong>of</strong> the protein and the<br />
solvent effects, like additional water uptake to the complex and partial protonation (see<br />
4.2.3., p. 73).<br />
In an effort to elucidate the structural features <strong>of</strong> <strong>mRNA</strong> 5' cap analogues responsible<br />
for the observed <strong>eIF4E</strong> binding energetics, thermodynamic investigations have been<br />
undertaken. The aim <strong>of</strong> the present chapter is to analyse the specific binding in terms <strong>of</strong><br />
quantitative thermodynamic parameters determined independently by the van't H<strong>of</strong>f<br />
method and isothermal titration calorimetry (ITC). Such quantitative data are <strong>of</strong> primary<br />
importance for rational design <strong>of</strong> new cap analogues <strong>of</strong> potential therapeutic activity since<br />
the high <strong>eIF4E</strong> cellular level is relevant to malignancy and apoptosis 13 .<br />
In this chapter the absolute temperature scale will be used, T (K) = t (°C) + 273.16.<br />
4.4.1. Van't H<strong>of</strong>f <strong>Analysis</strong> <strong>of</strong> Binding <strong>of</strong> <strong>eIF4E</strong> to Cap Analogues<br />
Fluorescence titration experiments were performed to obtain values <strong>of</strong> equilibrium<br />
association constants for nine structurally altered cap analogues, m 7 GMP, m 7 GDP, m 7 GTP,<br />
m3 2,2,7 GTP, bz 7 GTP, p-Cl-bz 7 GTP, m 7 GpppG, m 7 GpppC, and m 7 Gppppm 7 G in the<br />
temperature range <strong>of</strong> 280 – 314 K. Exemplary binding isotherms for <strong>eIF4E</strong> association<br />
<strong>with</strong> m 7 GpppG at six selected temperatures are presented in Fig. 4-29. The equilibrium<br />
association constants (Kas) and the corresponding standard molar free energies <strong>of</strong><br />
formation <strong>of</strong> the complexes (ΔG°) are gathered in Table 4-7.<br />
97
Table 4-7. Association constants (Kas ± SD) and standard molar free energies (ΔG° ± SD) for<br />
binding <strong>of</strong> selected mononucleotide and dinucleotide cap analogues to <strong>eIF4E</strong> at different<br />
temperatures (T), at pH 7.2.<br />
m 7 GMP<br />
T (K)<br />
279.0<br />
280.2<br />
283.9<br />
288.2<br />
293.2<br />
297.9<br />
301.2<br />
304.2<br />
306.3<br />
309.7<br />
313.2<br />
m 7 GDP<br />
T (K)<br />
280.2<br />
283.2<br />
286.2<br />
286.7<br />
287.7<br />
288.7<br />
289.2<br />
293.2<br />
297.8<br />
304.2<br />
309.7<br />
313.2<br />
⋅10 −6 (Μ −1 )<br />
Κ as ⋅10<br />
Y SD<br />
2.734 0.605<br />
2.492 0.201<br />
1.260 0.390<br />
1.430 0.090<br />
0.766 0.090<br />
0.870 0.160<br />
0.780 0.110<br />
0.475 0.157<br />
0.580 0.181<br />
0.473 0.250<br />
0.838 0.328<br />
⋅10 −6 (Μ −1 )<br />
Κ as ⋅10<br />
Y SD<br />
74.33 10.57<br />
74.58 6.75<br />
46.64 4.08<br />
38.25 3.36<br />
43.18 6.05<br />
38.57 4.16<br />
37.50 4.46<br />
20.40 1.54<br />
17.48 3.15<br />
10.48 1.32<br />
5.04 0.74<br />
6.00 1.70<br />
98<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-34.38 0.51<br />
-34.31 0.19<br />
-33.15 0.73<br />
-33.96 0.15<br />
-33.02 0.29<br />
-33.87 0.46<br />
-33.97 0.35<br />
-33.06 0.84<br />
-33.79 0.79<br />
-33.64 1.36<br />
-35.51 1.02<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-42.22 0.33<br />
-42.68 0.21<br />
-42.01 0.21<br />
-41.61 0.21<br />
-42.05 0.34<br />
-41.92 0.26<br />
-41.93 0.29<br />
-41.02 0.18<br />
-41.29 0.45<br />
-40.88 0.32<br />
-39.73 0.38<br />
-40.64 0.74
m 7 GTP<br />
T (K)<br />
281.7<br />
283.6<br />
286.0<br />
288.2<br />
290.6<br />
293.2<br />
295.1<br />
296.5<br />
297.9<br />
299.3<br />
301.2<br />
303.4<br />
305.2<br />
309.0<br />
311.4<br />
313.5<br />
m3 2,2,7 GTP<br />
T (K)<br />
281.3<br />
284.9<br />
286.2<br />
290.2<br />
293.0<br />
296.1<br />
298.6<br />
301.6<br />
304.4<br />
309.7<br />
313.2<br />
Kas ⋅ 10 -6 (M -1 )<br />
Y SD<br />
389.4 45.6<br />
333.5 157.4<br />
352.3 97.1<br />
161.4 35.7<br />
139.7 61.8<br />
116.4 13.0<br />
112.3 22.4<br />
105.3 16.8<br />
54.1 12.1<br />
50.7 10.0<br />
67.7 13.6<br />
44.6 6.8<br />
63.0 8.4<br />
28.5 7.2<br />
17.1 2.5<br />
16.6 9.3<br />
Kas ⋅ 10 -6 (M -1 )<br />
Y SD<br />
0.291 0.155<br />
0.175 0.098<br />
0.155 0.084<br />
0.129 0.110<br />
0.143 0.080<br />
0.112 0.145<br />
0.112 0.100<br />
0.138 0.113<br />
0.126 0.040<br />
0.209 0.087<br />
0.290 0.408<br />
99<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-46.32 0.27<br />
-46.27 1.11<br />
-46.79 0.66<br />
-45.28 0.53<br />
-45.31 1.07<br />
-45.47 0.25<br />
-45.47 0.49<br />
-45.53 0.39<br />
-44.10 0.55<br />
-44.14 0.49<br />
-45.15 0.50<br />
-44.43 0.38<br />
-45.56 0.34<br />
-44.10 0.65<br />
-43.11 0.37<br />
-43.32 1.47<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-29.42 1.24<br />
-28.60 1.32<br />
-28.44 1.29<br />
-28.38 2.07<br />
-28.92 1.36<br />
-28.62 3.18<br />
-28.85 2.23<br />
-29.68 2.04<br />
-29.72 0.80<br />
-31.54 1.07<br />
-32.75 3.67
z 7 GTP<br />
T (K)<br />
281.6<br />
289.1<br />
291.2<br />
293.2<br />
299.2<br />
304.2<br />
309.7<br />
313.2<br />
p-Cl-bz 7 GTP<br />
T (K)<br />
281.4<br />
283.7<br />
285.9<br />
287.4<br />
288.8<br />
290.8<br />
292.0<br />
293.2<br />
297.0<br />
301.6<br />
304.7<br />
308.5<br />
309.1<br />
312.0<br />
313.8<br />
Kas ⋅ 10 -6 (M -1 )<br />
Y SD<br />
58.60 6.80<br />
32.94 3.20<br />
24.20 1.90<br />
17.50 0.50<br />
12.88 0.40<br />
9.88 0.95<br />
7.46 1.03<br />
4.26 0.96<br />
Kas ⋅ 10 -6 (M -1 )<br />
Y SD<br />
91.2 13.2<br />
84.8 12.9<br />
80.0 12.4<br />
113.3 39.6<br />
57.3 4.6<br />
51.5 4.7<br />
85.0 9.8<br />
47.9 7.8<br />
76.6 17.4<br />
29.9 3.8<br />
32.4 4.1<br />
29.7 5.4<br />
16.4 3.5<br />
21.3 4.2<br />
19.7 4.0<br />
100<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-41.87 0.27<br />
-41.60 0.23<br />
-41.16 0.19<br />
-40.65 0.07<br />
-40.72 0.08<br />
-40.74 0.24<br />
-40.74 0.36<br />
-39.75 0.59<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-42.88 0.34<br />
-43.06 0.36<br />
-43.25 0.37<br />
-44.31 0.83<br />
-42.90 0.19<br />
-42.93 0.22<br />
-44.32 0.28<br />
-43.10 0.40<br />
-44.82 0.56<br />
-43.16 0.32<br />
-43.81 0.32<br />
-44.13 0.46<br />
-42.69 0.55<br />
-43.77 0.51<br />
-43.82 0.53
m 7 GpppG<br />
T (K)<br />
m 7 GpppC<br />
280.6<br />
283.9<br />
288.2<br />
293.2<br />
293.2<br />
297.6<br />
301.4<br />
304.9<br />
309.8<br />
312.6<br />
T (K)<br />
280.0<br />
284.3<br />
287.4<br />
290.2<br />
292.8<br />
296.6<br />
299.4<br />
304.0<br />
308.6<br />
313.1<br />
m 7 Gppppm 7 G<br />
T (K)<br />
281.4<br />
283.8<br />
287.8<br />
290.4<br />
292.7<br />
293.2<br />
296.8<br />
302.2<br />
304.9<br />
308.7<br />
312.6<br />
⋅10 −6 (Μ −1 )<br />
Κ as ⋅10<br />
Y SD<br />
24.94 1.05<br />
23.70 4.35<br />
12.94 1.20<br />
7.39 0.46<br />
6.50 0.50<br />
5.13 0.81<br />
4.01 0.61<br />
2.29 0.52<br />
2.41 0.27<br />
2.56 0.22<br />
⋅10 −6 (Μ −1 )<br />
Κ as ⋅10<br />
Y SD<br />
12.25 1.22<br />
8.11 0.82<br />
7.09 0.86<br />
5.16 0.64<br />
2.76 0.48<br />
3.39 0.52<br />
2.44 0.50<br />
2.42 0.43<br />
1.27 0.56<br />
2.74 1.80<br />
micro<br />
Kas ⋅10 -6 (M -1 )<br />
Y SD<br />
125.85 46.68<br />
75.00 20.50<br />
48.70 5.45<br />
35.70 5.20<br />
23.95 1.95<br />
23.48 2.40<br />
20.00 2.10<br />
10.72 1.10<br />
6.62 1.57<br />
5.88 1.07<br />
4.91 0.27<br />
101<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-39.73 0.10<br />
-40.08 0.43<br />
-39.23 0.22<br />
-38.55 0.15<br />
-38.24 0.19<br />
-38.22 0.39<br />
-38.10 0.38<br />
-37.12 0.58<br />
-37.84 0.29<br />
-38.35 0.22<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-37.99 0.23<br />
-37.60 0.24<br />
-37.69 0.29<br />
-37.29 0.30<br />
-36.10 0.42<br />
-37.07 0.38<br />
-36.61 0.51<br />
-37.15 0.45<br />
-36.06 1.13<br />
-38.58 1.71<br />
ΔG° (kJ/mol)<br />
Y SD<br />
-43.63 0.87<br />
-42.78 0.64<br />
-42.35 0.27<br />
-41.98 0.35<br />
-41.34 0.20<br />
-41.37 0.25<br />
-41.48 0.26<br />
-40.67 0.26<br />
-39.81 0.60<br />
-40.00 0.47<br />
-40.04 0.14
Fluorescence (a. u.)<br />
100<br />
80<br />
60<br />
40<br />
10 -3 10 -2 10 -1 10 0 10 1<br />
m 7 GpppG (μM)<br />
Figure 4-29. Quenching <strong>of</strong> <strong>eIF4E</strong> intrinsic fluorescence upon titration <strong>with</strong> 7-methylGpppG.<br />
Increasing fluorescence intensity at higher concentration <strong>of</strong> 7-methylGpppG originates from the<br />
free ligand in solution. Binding isotherms for different temperatures: 312.6 K, 304.9 K, 297.6 K,<br />
293.2 K, 288.2 K, 280.6 K. Titrations were performed in 50 mM Hepes/KOH (pH 7.2), 100 mM<br />
KCl, 1 mM DTT, 0.5 mM EDTA.<br />
The results <strong>of</strong> the fluorescence titrations have been analysed by the van't H<strong>of</strong>f<br />
method. For the symmetrical cap analogue, m 7 Gppppm 7 G, the microscopic association<br />
constant was taken into the analysis. Standard molar enthalpy changes (ΔH°) and standard<br />
molar entropy changes (ΔS°) at 293 K determined from the van’t H<strong>of</strong>f dependencies are<br />
collected in Table 4-8. The association <strong>of</strong> <strong>eIF4E</strong> to <strong>mRNA</strong> 5' cap at 273 K has been found<br />
to be generally enthalpy-driven for the whole series, and entropy-opposed or –driven,<br />
depending on the cap analogue (Fig. 4-30). Strong, specific interactions may be attributed<br />
to fairly high enthalpy <strong>of</strong> association, from −50 to −81 kJ⋅mol -1 , while for the less specific<br />
ligands ΔH° is −17 to −36 kJ⋅mol -1 . One exception among the most tightly binding<br />
analogues is p-Cl-bz 7 GTP that has a small binding enthalpy <strong>of</strong> only –38.5 kJ/mol but a<br />
large positive binding entropy <strong>of</strong> +16.7 J/mol⋅K. The binding entropy <strong>of</strong> the remaining cap<br />
analogues ranges from +40 to −136 J⋅mol -1 ⋅K -1 .<br />
The enthalpy change plays a more pronounced role for binding <strong>of</strong> the analogues <strong>with</strong><br />
longer phosphate chains, which is entropy-opposed. An enthalpy decrease that results from<br />
comparison <strong>of</strong> the binding enthalpies for m 7 GMP and m 7 GDP equals ΔΔH°m7GMPm7GDP =<br />
−25.9 ± 8.4 kJ/mol, and elongation <strong>of</strong> the phosphate chain to three members gives the<br />
further enthalpy decrease <strong>of</strong> ΔΔH°m7GDPm7GTP = −12.4 ± 4.6 kJ/mol. These enthalpy<br />
differences, when divided by the number <strong>of</strong> hydrogen bonds or salt bridges formed by the<br />
102<br />
312.6 K<br />
304.9 K<br />
297.6 K<br />
293.2 K<br />
288.2 K<br />
280.6 K
β and γ phosphate groups (2 and 1, respectively), yield almost equal values <strong>of</strong> the unitary<br />
enthalpy change per one bond, ΔΔH°B ≈ −12.8 ± 9.6 kJ/mol, which is a typical value. 240<br />
Although in general the binding entropy cannot be theoretically decomposed into<br />
individual atom-atom interactions, in this case the entropic penalties, that equal<br />
ΔΔS°m7GMPm7GDP = −61 ± 11 and ΔΔS°m7GDPm7GTP = −30 ± 16 J/mol⋅K, appear to yield<br />
an average <strong>of</strong> ΔΔS°B ≈ −30 ± 19 J/mol⋅K per bond. This observation can serve as an<br />
indicator that binding <strong>of</strong> the subsequent phosphate group is approximately independent<br />
from binding <strong>of</strong> the previous one, i. e. there is no visible effect associated <strong>with</strong> linkage <strong>of</strong><br />
the phosphates, arising from a change in the number <strong>of</strong> degrees <strong>of</strong> freedom.<br />
The binding entropy is more conducive to association <strong>of</strong> the analogues possessing<br />
more or larger substituents at the guanine moiety. For the larger and larger substituents in<br />
the series <strong>of</strong> triphosphates: 7-methyl-, 7-benzyl, 7-para-chloro-benzylGTP, the ΔS° value<br />
increases subsequently by +46 ± 18 and +69 ± 20 J/mol⋅K. This is caused most likely by<br />
expulsion <strong>of</strong> several water molecules from the depth <strong>of</strong> the cap-binding slot into the bulk<br />
solvent, proportionally to the N(7)-substituent volume. The large and electronegative<br />
chloride atom at the N 7 -benzyl ring can attenuate the van der Waals interaction <strong>with</strong> Trp-<br />
166 and destroy the specific water network inside the <strong>eIF4E</strong> cap-binding centre due to<br />
steric and electrostatic effects (Fig. 4-13, p. 67). Consequently, the binding enthalpy for p-<br />
Cl-bz 7 GTP is significantly less negative than that for bz 7 GTP by ~ 18 kJ/mol. The higher<br />
affinity <strong>of</strong> p-Cl- bz 7 GTP for <strong>eIF4E</strong> arises from the more favourable entropy change.<br />
ΔΔH°° (kJ/mol)<br />
50<br />
0<br />
-50<br />
-100<br />
-150<br />
-45 -40 -35 -30<br />
ΔG° (kJ/mol)<br />
Figure 4-30. Enthalpic (ΔH°) and entropic (−TΔS°) contributions to Gibbs free energy (ΔG°) <strong>of</strong><br />
<strong>eIF4E</strong> binding to structurally different cap analogues at 293 K.<br />
103<br />
-TΔ S°° (kJ/mol)<br />
50<br />
0<br />
-50<br />
-100<br />
-150<br />
-45 -40 -35 -30<br />
ΔG° (kJ/mol)
m 7 , 2 , 2<br />
3<br />
In the case <strong>of</strong> GTP , the relative contribution <strong>of</strong> the hydrophobic interactions to<br />
ΔG° increases due to fewer hydrogen bonds stabilizing the complex (see 4.2.2., p. 64) and<br />
much weaker cation-π stacking 241 which itself would provide a large enthalpic component<br />
(discussed below, 4.4.9.3., p. 124). Hence, the thermodynamic driving force for GTP<br />
becomes more entropic.<br />
104<br />
m 7 , 2 , 2<br />
3<br />
Table 4-8. Standard molar enthalpy changes (ΔH°) and entropy changes (ΔS°) at 293 K obtained<br />
from the van't H<strong>of</strong>f isobaric equation for binding <strong>of</strong> <strong>eIF4E</strong> to selected <strong>mRNA</strong> 5' cap analogues.<br />
Standard molar Gibbs free energy changes (ΔG°) calculated from the association constants at 392<br />
K are shown for reference, pH 7.2.<br />
Cap analogue ΔH° ΔS° ΔG°<br />
(kJ·mol -1 ) (J·mol -1 ·K -1 ) (kJ·mol -1 )<br />
m 7 GMP a<br />
m 7 GDP b<br />
m 7 GTP b<br />
bz 7 GTP b<br />
p-Cl-bz 7 GTP b<br />
m3 2,2,7 GTP a<br />
m 7 Gpppp(m 7 G)<br />
m 7 GpppG a<br />
m 7 GpppC a<br />
a, c<br />
-36.0 ± 7.9 -9.3 ± 3.4 -33.15 ± 0.20<br />
-61.9 ± 2.9 -69.8 ± 10.5 -41.031 ± 0.179<br />
-74.3 ± 3.6 -98.7 ± 12.1 -45.109 ± 0.090<br />
-56.5 ± 3.8 -52.7 ± 13.0 -40.669 ± 0.060<br />
-38.5 ± 4.6 +16.7 ± 15.5 -42.94 ± 0.21<br />
-16.6 ± 2.5 +40.3 ± 12.7 -28.94 ± 1.36<br />
-81 ± 54 -136 ± 88 -41.377 ± 0.146<br />
-65 ± 31 -91 ± 58 -38.555 ± 0.152<br />
-50 ± 28 -45 ± 29 -36.42 ± 0.40<br />
a temperature-dependent ΔH° and ΔS° (non-linear van’t H<strong>of</strong>f plot)<br />
b constant ΔH° and ΔS° (deviation <strong>of</strong> van’t H<strong>of</strong>f plot from linearity not detected, P(ν1, ν2) > 0.6)<br />
c the microscopic association constant (Kas micro ) has been taken into account<br />
Careful examination <strong>of</strong> the van’t H<strong>of</strong>f plots revealed that the linear character is lost<br />
for the analogues <strong>of</strong> moderate affinity for <strong>eIF4E</strong> (Fig. 4-31). The thermodynamic<br />
parameters that are related to the non-linearity, i. e. standard heat capacity change under<br />
constant pressure (ΔC°p), and the critical temperatures where ΔH° = 0 and ΔS° = 0 (TH and<br />
TS, respectively) are gathered in Table 4-9. The large<br />
o<br />
Δ C p values resulting from the non-<br />
linearity are surprisingly positive, from +1.66 up to +5.12 kJ⋅mol -1 ⋅K -1 for the m3 2,2,7 GTP -<br />
<strong>eIF4E</strong> association, and TH is higher than TS. The results derived from the non-linear fitting<br />
are statistically unambiguously superior to those derived from the linear one on the<br />
significance level (P) resulting from the Snedecor's F-test, from 0.049 to even less than<br />
0.0001. Involvement <strong>of</strong> the next fitting parameter that was related to a possible linear<br />
dependence <strong>of</strong> ΔC°p on temperature did not render further improvement <strong>of</strong> the results.
ln Kas<br />
ln Kas<br />
22<br />
20<br />
18<br />
16<br />
14<br />
12<br />
10<br />
22<br />
20<br />
18<br />
16<br />
14<br />
12<br />
10<br />
m 7 GTP<br />
m 7 GDP<br />
m 7 GMP<br />
m 7 m<br />
GpppC<br />
7 m<br />
GpppG<br />
7 Gppppm 7 G<br />
3.2 3.3 3.4 3.5 3.6<br />
T -1 ⋅ 10 3 (K -1 )<br />
(a)<br />
(c)<br />
105<br />
bz 7 p-Cl-bz<br />
GTP<br />
7 GTP<br />
2,2,7<br />
m3 GTP<br />
3.2 3.3 3.4 3.5 3.6<br />
T -1 ⋅ 10 3 (K -1 )<br />
(b)<br />
Figure 4-31. Dependence <strong>of</strong> van’t H<strong>of</strong>f<br />
plots for <strong>eIF4E</strong> - cap binding on structural<br />
alterations <strong>of</strong> the cap analogue:<br />
(a) elongation <strong>of</strong> the phosphate chain,<br />
(b) replacement <strong>of</strong> the 7-methyl group for<br />
larger substituents and dimethylation <strong>of</strong> 2amino<br />
group,<br />
(c) change <strong>of</strong> the second base and number <strong>of</strong><br />
the phosphate groups.
Table 4-9. Critical temperatures TH (where ΔH° = 0), TS (ΔS° = 0), and standard molar heat<br />
capacity changes under constant pressure (ΔCp°) obtained from fitting <strong>of</strong> the non-linear van't H<strong>of</strong>f<br />
equation to the equilibrium association constants for selected <strong>mRNA</strong> 5' cap analogues, at pH 7.2.<br />
The relative decrease in sum-<strong>of</strong>-squares (F) for the number <strong>of</strong> degrees <strong>of</strong> freedom (ν2) in<br />
comparison <strong>with</strong> that for the linear model (ν1), and the probability <strong>of</strong> random improvement <strong>of</strong><br />
goodness <strong>of</strong> fit P(ν1, ν2). Calculated Gibbs free energies at TS, ΔG°TS = ΔH°TS, and at TH, ΔG°TH =<br />
−TH⋅ΔS°TH.<br />
Cap analogue TH TS ΔCp° F P(ν1, ν2)<br />
(K) (K) (kJ·mol -1 ·K -1 )<br />
m 7 Gpppp(m 7 G) a 342.0 ± 16.0 318.0 ± 7.8 +1.66 ± 0.57 8.54 0.019<br />
m 7 GpppG 327.1 ± 15.2 307.4 ± 6.0 +1.92 ± 0.93 6.36 0.040<br />
m 7 GpppC 310.0 ± 6.2 297.6 ± 2.1 +2.96 ± 1.25 5.65 0.049<br />
m 7 GMP 306.9 ± 4.8 294.20 ± 1.68 +2.62 ± 0.97 7.34 0.027<br />
m3 2,2,7 GTP 296.41 ± 0.43 290.86 ± 0.69 +5.12 ± 0.48 115 TH, which is most <strong>of</strong>ten<br />
attributed to burial <strong>of</strong> solvent accessible hydrophobic molecular surface upon complex<br />
formation. 131<br />
To confirm validity <strong>of</strong> the exceptional results derived from the van't H<strong>of</strong>f method for<br />
interaction <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the cap analogues, direct calorimetric measurements have been<br />
o carried out. The standard enthalpy changes ( Δ Hcal<br />
) have been determined for one selected<br />
cap analogue, m 7 GpppG, at four different temperatures by means <strong>of</strong> Isothermal Titration<br />
Calorimetry (ITC) (Fig. 4-32). In order to control the activity <strong>of</strong> <strong>eIF4E</strong> during the ITC<br />
106
experiment, concurrent fluorometric titrations have been run <strong>with</strong> use <strong>of</strong> the same protein<br />
samples, incubated at a given temperature for the same time. The synchronised<br />
measurements <strong>of</strong> the protein intrinsic fluorescence quenching allowed to determine the<br />
actual concentration <strong>of</strong> the active protein ([Pact]) at each temperature. [Pact] at 288.1, 293.1,<br />
298.4, and 303.2 K was 8.97, 7.42, 3.61, and 5.35 μM, respectively. The enthalpy<br />
estimates for the assumed 100 % protein activity (10 μM), and those corrected for the<br />
active protein concentration determined from fluorometric titrations, are shown in<br />
comparison <strong>with</strong> the van't H<strong>of</strong>f enthalpy change (Fig. 4-33). The corrected calorimetric<br />
enthalpies yield a slope <strong>of</strong><br />
the result <strong>of</strong> the van't H<strong>of</strong>f analysis (<br />
o<br />
Δ C p = +1.966 ± 0.061 kJ⋅mol -1 K -1 (Table 4-10), which confirms<br />
o<br />
Δ C p = +1.92 ± 0.93 kJ⋅mol -1 K -1 ).<br />
Table 4-10. The van't H<strong>of</strong>f and calorimetric thermodynamic parameters for binding <strong>of</strong> m 7 GpppG to<br />
<strong>eIF4E</strong> in Hepes buffer at pH 7.2<br />
ΔH<br />
ΔH<br />
o a<br />
cal<br />
o b<br />
ion<br />
o<br />
cal ion<br />
d<br />
ΔH<br />
−<br />
o<br />
ΔH<br />
vH<br />
o<br />
ΔS e<br />
Temperature (K)<br />
288.1 293.1 298.4 303.2 ΔCp°(kJ⋅mol -1 K -1 )<br />
Enthalpy change (kJ⋅mol -1 )<br />
−65.5 ± 3.2 −54.4 ± 4.0 −47.3 ± 8.4 −35.5 ± 1.7 +1.966 ± 0.061 f<br />
+8.53 +8.66 +8.81 +8.94 +0.0273 ± 0.0003 f<br />
c −74.0 ± 3.2 −63.1 ± 4.0 −56.1 ± 8.4 −44.4 ± 1.7 +1.941 ± 0.059 f<br />
−75 ± 36 −65 ± 31 −56 ± 26 −46 ± 22 +1.92 ± 0.93 g<br />
Entropy change (J⋅mol -1 ⋅K -1 )<br />
−124 ± 71 −91 ± 58 −57 ± 47 −26 ± 40<br />
a<br />
directly measured total enthalpy changes<br />
b o<br />
buffer ionization heats at pH 7.2; estimated δ Δ Hion<br />
= ± 0.09<br />
c o<br />
o<br />
net calorimetric enthalpy changes, Δ Hcal−ion = Δ Hcal<br />
− ΔH<br />
d van't H<strong>of</strong>f enthalpy changes<br />
e entropy changes from the van't H<strong>of</strong>f method<br />
f calculated from linear temperature-dependence <strong>of</strong> corresponding<br />
g calculated from non-linear van't H<strong>of</strong>f dependence<br />
107<br />
o<br />
ion<br />
o<br />
ΔH<br />
x
Power ( μ J⋅ s -1 )<br />
0<br />
-2<br />
-4<br />
-6<br />
-8<br />
-10<br />
-12<br />
-14<br />
0<br />
-2<br />
-4<br />
-6<br />
-8<br />
-10<br />
-12<br />
-14<br />
Time (s)<br />
Time (s)<br />
Figure 4-32. Modified "single injection" ITC curves at 288.1 K (left panels) and 303.2 K (right<br />
panels). Each experiment consisted <strong>of</strong> a main 40 μl injection, preceded by two 1 μl injections<br />
enabling to calculate a correction for the instrumental artifacts, and followed by two 4 μl injections<br />
to check the protein saturation <strong>with</strong> the ligand. Measured heat <strong>of</strong> mixing <strong>of</strong> 7-methylGpppG <strong>with</strong><br />
<strong>eIF4E</strong> solution (—,—), and <strong>of</strong> 7-methylGpppG dilution in buffer (—,—) (upper panels). Calculated<br />
reaction heat <strong>of</strong> binding <strong>of</strong> 7-methylGpppG to <strong>eIF4E</strong> at pH 7.2 in Hepes buffer (—) (bottom<br />
panels).<br />
4.4.3. Protonation Equilibrium <strong>of</strong> m 7 GpppG<br />
o A systematic positive shift <strong>of</strong> the calorimetric enthalpies ( Δ Hcal<br />
) in comparison <strong>with</strong><br />
o their van't H<strong>of</strong>f counterparts ( Δ H vH ) appears from the data (Fig. 4-33, Table 4-10).<br />
Although the numerical uncertainty <strong>of</strong><br />
the difference between<br />
288.1 K<br />
500 1000 1500<br />
o<br />
Δ H vH is about ± 30 kJ⋅mol -1 , the average value <strong>of</strong><br />
o o<br />
Δ Hcal<br />
and Δ H vH <strong>of</strong> about +9.7 kJ⋅mol -1 seems to be well specified.<br />
Differences between the calorimetric and the van't H<strong>of</strong>f enthalpy estimates were the<br />
subject <strong>of</strong> empirical and theoretical analyses for various association processes 244-250 . The<br />
observed discrepancies were ascribed to contributions <strong>of</strong> usually unknown molecular<br />
108<br />
303.2 K<br />
500 1000 1500
Δ H° cal (kJ ⋅mol -1 )<br />
Figure 4-33. Total calorimetric enthalpies corrected for ligand dilution, assuming 100 % protein<br />
activity ( ,------), and those corrected also for active protein concentration, ( ,——). Temperature<br />
dependence <strong>of</strong> the van't H<strong>of</strong>f enthalpy is shown for comparison ( ).<br />
transitions or coupled processes, other than the net complex formation, to<br />
erroneous apparent values <strong>of</strong><br />
0<br />
-20<br />
-40<br />
-60<br />
-80<br />
283 288 293 298 303 308<br />
o<br />
Δ H vH and<br />
Temperature (K)<br />
109<br />
o<br />
p<br />
o<br />
cal<br />
Δ H , and/or<br />
Δ C , arising from the experimental noise. In the<br />
case <strong>of</strong> cap − <strong>eIF4E</strong> binding the latter obscuring effect is eliminated, as testified by the<br />
accordance <strong>of</strong> the van't H<strong>of</strong>f and calorimetric<br />
o<br />
p<br />
Δ C values. This approximately constant<br />
difference can be analysed in terms <strong>of</strong> the protonation equilibria 251 . 7-methylGpppG exists<br />
as a mixture <strong>of</strong> cationic (58%) and zwitterionic (42%) forms at pH 7.2 (pKa = 7.35 ± 0.05<br />
for the N(1)-H proton <strong>of</strong> 7-methylguanine moiety 177 , Scheme 3-1, p. 23). Several<br />
contradictory conclusions were reported regarding the ionic state <strong>of</strong> the <strong>mRNA</strong> 5' cap that<br />
binds to <strong>eIF4E</strong> most tightly. Initially, the zwitterionic form <strong>of</strong> 7-methylguanine was<br />
postulated to interact <strong>with</strong> <strong>eIF4E</strong> 155,156 . In contrast, the crystallographic and NMR<br />
structures revealed the spatial distances suitable for formation <strong>of</strong> a hydrogen bond between<br />
Glu103 and N(1)-H <strong>of</strong> 7-methylguanine 1,48,75,76 , pointing to the cationic form <strong>of</strong> 7-<br />
methylguanine. This hydrogen bond is also present in the ternary 7-methylGDP − <strong>eIF4E</strong> −<br />
eIF4GII peptide complex at pH 8.5 91 . However, the N(1)-H proton has not been directly<br />
observed by NMR 76 . The binding studies as a function <strong>of</strong> pH showed the upward shift <strong>of</strong><br />
pKa <strong>of</strong> N(1)-H inside the <strong>eIF4E</strong> binding site, suggesting that the tightest binding is<br />
accomplished through the cationic form <strong>of</strong> 7-methylguanosine (see 4.2.3.2., p. 83). The<br />
<strong>eIF4E</strong> − cap association that is accompanied by the partial protonation <strong>of</strong> the ligand must<br />
be equilibrated by additional deprotonation <strong>of</strong> the buffer to keep the cation-zwitterion<br />
equilibrium <strong>of</strong> the free ligand at constant pH. As shown in Table 4-10, the calculated
o<br />
ion<br />
contribution <strong>of</strong> the Hepes ionization heat ( Δ H ) to the total reaction heat is in a very good<br />
agreement <strong>with</strong> the difference between<br />
o<br />
cal<br />
Δ H and<br />
110<br />
o<br />
Δ H vH . This demonstrates the linkage<br />
between 7-methylGpppG − <strong>eIF4E</strong> binding and concomitant protonation <strong>of</strong> the residue<br />
which has pKa = 7.35 in the unbound state. Taking into account the net calorimetric<br />
enthalpy changes related to cap binding by <strong>eIF4E</strong>,<br />
o<br />
cal ion<br />
Δ H − = Δ Hcal<br />
− Δ Hion<br />
,<br />
o<br />
o<br />
the calorimetric heat capacity change corrected for buffer ionization is<br />
0.059 kJ⋅mol -1 K -1 .<br />
o<br />
p<br />
Δ C = +1.941 ±<br />
The coupling <strong>of</strong> the binding process <strong>with</strong> the acidic-basic equilibrium <strong>of</strong> the ligand is<br />
nonmandatory 122 , since the zwitterionic form <strong>of</strong> 7-methylGpppG is also able to bind to the<br />
protein via weaker interactions <strong>of</strong> 7-methylG moiety and almost unchanged interactions <strong>of</strong><br />
the phosphate chain. It was shown that the protein-induced shift in the two-state transition<br />
<strong>of</strong> the ligand could contribute to the observed heat capacity change 122 . This contribution<br />
may be either positive or negative, depending on the unknown values <strong>of</strong> the intrinsic<br />
enthalpy changes and equilibrium constants that describe binding <strong>of</strong> the cationic and the<br />
zwitterionic forms <strong>of</strong> 7-methylGpppG to <strong>eIF4E</strong> independently.<br />
4.4.4. Conformational Equilibrium <strong>of</strong> Cap Analogues<br />
Association <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the <strong>mRNA</strong> 5' terminus is also directly coupled <strong>with</strong><br />
intramolecular self-stacking <strong>of</strong> the cap. The question arises whether and to how extent this<br />
coupled process influences binding to <strong>eIF4E</strong>. Self-stacking provides for additional<br />
o o enthalpic ( Δ Hsst<br />
) and entropic ( Ssst<br />
o psst<br />
C<br />
Δ ) contributions, and an apparent molar heat capacity<br />
change ( Δ ), resulting from an induced shift in the conformational equilibrium <strong>of</strong> the<br />
ligand upon binding to the protein. 122 As only the unstacked form <strong>of</strong> dinucleotide cap<br />
analogues can penetrate the <strong>eIF4E</strong> cap-binding slot, the coupling is mandatory.<br />
The known thermodynamic parameters describing self-stacking <strong>of</strong> the cationic form<br />
(pH 5.2) and zwitterionic form (pH 9.0) <strong>of</strong> some dinucleotide cap analogues at 293 K 198<br />
gave a possibility to assess the influence <strong>of</strong> this coupled process on the cap − <strong>eIF4E</strong><br />
complex formation. On the basis <strong>of</strong> these parameters, required values <strong>of</strong> equilibrium<br />
constants (1K) and standard molar enthalpy changes (1ΔH°) for self-stacking at pH 7.2 have<br />
been calculated, and next the contributions from self-stacking to the observed<br />
thermodynamic parameters have been determined (Table 4-11). Both<br />
o o<br />
Δ Hsst<br />
and Ssst<br />
Δ for
each dinucleotide analogue are positive and relatively large. They reduce the negative<br />
values <strong>of</strong> the intrinsic thermodynamic parameters ( Δ H , Δ S , Table 4-12) to a substantial<br />
extent, leading to the apparently less negative values <strong>of</strong> the observed ΔH°cal-ion, ΔH°vH and<br />
ΔS°, as reported above (Table 4-8). In case <strong>of</strong> m 7 GpppG, the Δ H and Δ S values change<br />
by 12 – 17 %, and even by 17 – 46 %, respectively, depending on temperature.<br />
Table 4-11. Equilibrium constants (1K) and standard molar enthalpy changes (1ΔH°) for<br />
intramolecular self-stacking <strong>of</strong> dinucleotide cap analogues; and apparent contributions to standard<br />
molar enthalpy (ΔH°sst), entropy (ΔS°sst), and heat capacity (ΔCp°sst) changes <strong>of</strong> <strong>eIF4E</strong> association<br />
<strong>with</strong> cap analogues, resulting from the self-stacking equilibrium shift that is induced by the<br />
mandatory coupling <strong>of</strong> unstacking to the binding, at 293 K.<br />
Cap analogue 1K 1ΔH° ΔH°sst ΔS°sst ΔCp°sst<br />
(kJ·mol -1 ) (kJ·mol -1 ) (J·mol -1 ·K -1 ) (J·mol -1 ·K -1 )<br />
pH 5.2 a<br />
m 7 GpppG 1.40 -15.2 ± 0.5<br />
m 7 GpppC<br />
pH 9.0<br />
0.18 -12.0 ± 0.8<br />
a<br />
m 7 GpppG 1.58 -18.6 ± 0.7<br />
m 7 Gppp(m 7 G)<br />
pH 7.2<br />
0.48 -21.1 ± 0.9<br />
m 7 GpppG b<br />
111<br />
o<br />
0<br />
1.47 -16.6 ± 0.4 +9.89 ± 1.17 +30.5 ± 2.6 -93.2 ± 10.4<br />
m 7 Gppp(m 7 G) c 0.32 -15.8 ± 0.7 +3.85 ± 0.59 +10.81 ± 1.38 -64.5 ± 8.3<br />
m 7 GpppC d<br />
0.18 -12.0 ± 0.8 +1.83 ± 0.44 +4.87 ± 0.82 -26.1 ± 5.8<br />
a data from 198<br />
b values calculated from pKa = 7.35 ± 0.05 177 , 1K and 1ΔH° at pH 5.2 and 9.0<br />
c values estimated from the assumptions: pKa = 7.2 for both ends, no stacking for the double<br />
cationic form, stacking for the zwitterionic-cationic form the same as for the double zwitterionic<br />
form (pH 9.0)<br />
d values estimated from the assumptions: 1K and 1ΔH° the same as for the cationic form (pH 5.2)<br />
Table 4-12. Calculated standard molar enthalpy (ΔH°0), entropy (ΔS°0), and Gibbs free energy<br />
changes (ΔG°0) at 293 K, pH 7.2, for intrinsic binding <strong>of</strong> the unstacked dinucleotide cap analogues<br />
to <strong>eIF4E</strong>, at 293 K, pH 7.2.<br />
Cap analogue ΔH°0 ΔS°0 ΔG°0<br />
(kJ·mol -1 ) (J·mol -1 ·K -1 ) (kJ·mol -1 )<br />
m 7 Gpppp(m 7 G) b<br />
-85 ± 54 -147 ± 88 -42 ± 60<br />
m 7 GpppG -75 ± 31 -122 ± 58 -39 ± 35<br />
m 7 GpppC -52 ± 28 -50 ± 29 -37 ± 29<br />
b microscopic association constant has been taken into account<br />
o<br />
0<br />
o<br />
0<br />
o<br />
0
Table 4-13. Calculated critical temperatures TH 0 and TS 0 (where ΔH°0 = 0 and ΔS°0 = 0,<br />
respectively) and standard molar heat capacity changes under constant pressure (ΔCp°0) for the<br />
intrinsic binding <strong>of</strong> <strong>eIF4E</strong> to the unstacked dinucleotide cap analogues, at pH 7.2.<br />
Cap analogue TH 0 TS 0 ΔCp°0<br />
(K) (K) (kJ·mol -1 ·K -1 )<br />
m 7 Gpppp(m 7 G) a<br />
342 ± 35 319.2 ± 18.5 +1.73 ± 0.57<br />
m 7 GpppG 330 ± 23 311.5 ± 12.5 +2.01 ± 0.93<br />
m 7 GpppC 310.6 ± 11.8 298.1 ± 3.6 +2.99 ± 1.25<br />
a microscopic association constant has been taken into account<br />
While enthalpy and entropy changes <strong>of</strong> binding <strong>of</strong> the dinucleotide cap analogues to<br />
<strong>eIF4E</strong> are pr<strong>of</strong>oundly affected by the coupling between the binding and the unstacking, the<br />
intrinsic binding free energy for the unstacked caps ( Δ G , Table 4-12) are only negligibly<br />
enhanced in comparison <strong>with</strong> the resultant ΔG° values (constant <strong>with</strong>in SD). The negative<br />
o<br />
Δ C psst contributions to the resultant heat capacity changes are very small and almost<br />
temperature-independent. Assuming constant<br />
112<br />
o<br />
0<br />
o<br />
Δ C psst values, they encompass a range from<br />
–26 to −93 J⋅mol -1 K -1 for the three analogues (Table 4-11), and shift negligibly the heat<br />
capacity changes <strong>of</strong> the overall association <strong>with</strong> <strong>eIF4E</strong> from the intrinsic values for the<br />
o<br />
p0<br />
unstacked caps ( Δ C , Table 4-13) to the resultant values reported in Table 4-9. The<br />
critical temperatures TS0 and TH0 are also not significantly changed. Although the<br />
indirectly determined contributions from self-stacking as well as the intrinsic<br />
thermodynamic parameters for <strong>eIF4E</strong> binding to the unstacked cap are charged by<br />
significant uncertainties, they can serve as general indicators whether and how the complex<br />
formation is influenced by the coupled equilibrium <strong>of</strong> cap self-stacking.<br />
The determined small<br />
o<br />
Δ C psst values are very close to a value found as a contribution<br />
due to the coupling <strong>of</strong> adenine base unstacking to binding between single-stranded<br />
dA(pA)34 and the E. coli SSB protein, −62.8 ± 2.5 J⋅mol -1 K -1 per one stack 252 . This<br />
suggests that unstacking <strong>of</strong> the dinucleotide <strong>mRNA</strong> 5' cap analogue, i.e. between 7-<br />
methylguanosine and guanosine moieties linked via the symmetric 5'-5' triphosphate<br />
bridge, can be energetically considered in a similar way as oligodeoxyadenylates<br />
unstacking that accompanies specific binding <strong>of</strong> proteins to single-stranded DNA.
4.4.5. Could the Positive Values <strong>of</strong> ΔCp° Result from <strong>Protein</strong><br />
Deaggregation ?<br />
Two <strong>eIF4E</strong> (28-217) molecules are sticked together through the 4E-BP and eIF4G<br />
binding hydrophobic dorsal surface in the crystallographic asymmetric unit. 75,91 On the<br />
other hand, binding <strong>of</strong> cap forces the <strong>eIF4E</strong> (33-217) deaggregation (see 4.2.3.1.6.1., p.<br />
82). Therefore, some additional non-polar surface area would be exposed to water due to<br />
the binding-induced deaggregation. One could thus suspect that the positive<br />
113<br />
o<br />
p<br />
Δ C is related<br />
only to this side effect. Hence, the question arises whether the possible <strong>eIF4E</strong> (28-217)<br />
aggregation – deaggregation equilibrium coupled to the cap binding could be responsible<br />
for the observed positive heat capacity changes.<br />
The <strong>eIF4E</strong> (28-217) protein was filtered through 100 kD pore size immediately<br />
before experiments, and the probability <strong>of</strong> appearance <strong>of</strong> dimers in a dilute <strong>eIF4E</strong> solution<br />
was minimal. DLS measurements <strong>of</strong> this apo-<strong>eIF4E</strong> (28-217) showed that it was<br />
monodispersed. 253 Moreover, the main argument against this hypothesis is that then the<br />
putative deaggregation-related heat capacity change should be maximal for the strongest<br />
binding analogue (m 7 GTP), for which the deaggregation was the most effective. The<br />
situation is entirely opposite, since m 7 GTP and the other most tightly binding compounds<br />
yield<br />
o<br />
p<br />
Δ C = 0. Hence, it's impossible that the phenomenon which was independently<br />
detected by means <strong>of</strong> both emission spectroscopy and calorimetry at the <strong>eIF4E</strong><br />
concentration <strong>of</strong> ~ 0.1 μM and ~ 10 μM, respectively, could be a trivial one, resulting from<br />
a series <strong>of</strong> systematic experimental mistakes and wrong interpretation.<br />
4.4.6. Enthalpy-Entropy Compensation for Individual Cap Analogues<br />
Having verified the surprisingly positive and unusually high value <strong>of</strong> the heat<br />
capacity change for m 7 GpppG, one can ask about its consequences regarding the<br />
mechanism <strong>of</strong> cap – <strong>eIF4E</strong> interaction. As a result <strong>of</strong> the non-zero values <strong>of</strong><br />
o<br />
p<br />
Δ C , which<br />
are large in comparison <strong>with</strong> ΔS°, temperature-dependent enthalpy-entropy compensation<br />
occurs, i.e. the values <strong>of</strong> both the linear<br />
o<br />
Δ H vH term and the logarithmic TΔS° term in the<br />
expression for ΔG° increase <strong>with</strong> temperature, <strong>with</strong> nearly identical slopes (Fig. 4-<br />
34). 118,196 The large positive<br />
o<br />
p<br />
ΔC values for the <strong>eIF4E</strong> − cap association cause that the<br />
binding is enthalpy-driven at temperatures below TS (Table 4-9), while the entropy is
unfavourable, confirming the importance <strong>of</strong> electrostatic stabilization <strong>of</strong> the complex.<br />
Then, the interaction changes its thermodynamic character to both enthalpy- and entropy-<br />
driven between TS and TH, and finally, above TH the association becomes entropy-driven<br />
and enthalpy-opposed.<br />
Although the ΔG° function attains its maximum at TS, the stabilization <strong>of</strong> the<br />
complex does not decrease rapidly but is still efficient. For the two analogues which are <strong>of</strong><br />
frequent occurrence as natural <strong>mRNA</strong> 5' termini, m 7 GpppG and m 7 GpppC, the maximal<br />
values are about −38 and −37 kJ⋅mol -1 , respectively (Table 4-9). The fact <strong>of</strong> occurrence <strong>of</strong><br />
the non-zero heat capacity changes for the two natural dinucleotide cap analogues has three<br />
salient consequences:<br />
1. the interaction <strong>with</strong> <strong>eIF4E</strong> is both enthalpy and entropy favourable at biological<br />
temperatures (~ 310 K, 37 °C);<br />
2. the affinities <strong>of</strong> the two analogues for <strong>eIF4E</strong> are almost equal at ~ 310 K;<br />
3. the free energies <strong>of</strong> the complexes stabilization are relatively temperature-invariant<br />
over this range.<br />
On the contrary, the results obtained for the mononucleotide triphosphates show that<br />
thermodynamic driving forces make a distinction between the monomethylguanosine cap<br />
(m 7 GTP) and the trimethylguanosine cap ( m GTP<br />
7 , 2 , 2<br />
3<br />
114<br />
) at the biological temperature range.<br />
The association constant <strong>of</strong> the former is ~ 110-fold greater than that <strong>of</strong> the latter at 310 K<br />
(37 °C). The temperature-dependent thermodynamic parameters for m GTP<br />
7 , 2 , 2<br />
3 are ΔH°310K<br />
= +69.6 ± 6.9 kJ/mol and ΔS°310 K = +326 ± 33 J/mol⋅K. It is worth noting that due to the<br />
positive heat capacity change the m GTP<br />
7 , 2 , 2<br />
3 binding enthalpy attains the same absolute<br />
value as that for m 7 GTP but <strong>with</strong> the opposite sign, in spite <strong>of</strong> the fact that both analogues<br />
have the charged triphosphate chains. Hence, elevation <strong>of</strong> temperature at this range causes<br />
an increase <strong>of</strong> the <strong>eIF4E</strong> affinity for m GTP<br />
7 , 2 , 2<br />
3 and an affinity decrease for m 7 GTP. This<br />
phenomenon diminishes the predominance <strong>of</strong> m 7 GTP affinity in relation to m GTP<br />
7 , 2 , 2<br />
value <strong>of</strong> ~ 57 already at 313 K (40 °C).<br />
3 to a
Contributions to ΔΔG°° (kJ⋅⋅mol -1 )<br />
Contributions to ΔΔG o (kJ⋅mol -1 )<br />
20<br />
-30<br />
-40<br />
-50<br />
-60<br />
-70<br />
-80<br />
0<br />
-20<br />
-40<br />
-60<br />
-80<br />
-100<br />
TΔS°<br />
m 7 GpppG<br />
ΔH° cal-ion<br />
m 7 GTP<br />
ΔG o<br />
ΔH o<br />
T S<br />
TΔS o<br />
ΔG°<br />
280 290 300 310 320 330<br />
Temperature (K)<br />
ΔH° vH<br />
280 290 300 310 320 330<br />
Temperature (K)<br />
T H<br />
Figure 4-34. Temperature-dependent enthalpy-entropy compensation that accompanies the binding<br />
<strong>of</strong> m 7 GpppG, m 7 GpppC, and m3 2,2,7 GTP to <strong>eIF4E</strong>. Theoretical non-linear fit (—) to the binding free<br />
energies ΔG° (, , ), the entropic (− − −), and the van't H<strong>of</strong>f enthalpic (----) contributions to<br />
ΔG° as well as the calorimetric enthalpies corrected for ligand dilution, protein activity and buffer<br />
ionization heat ( ) <strong>with</strong> the linear regression (—) are plotted as a function <strong>of</strong> temperature. ΔS° = 0<br />
at TS and ΔH° = 0 at TH. Linear contributions to ΔG° <strong>of</strong> m 7 GTP () are shown for comparison.<br />
115<br />
Contributions to ΔG o (kJ⋅mol -1 )<br />
Contributions to ΔG o (kJ⋅mol -1 )<br />
60<br />
30<br />
0<br />
-30<br />
-60<br />
-90<br />
150<br />
100<br />
50<br />
0<br />
-50<br />
-100<br />
m 7 GpppC<br />
TΔS o<br />
ΔG o<br />
ΔH o<br />
280 290 300 310 320 330<br />
Temperature (K)<br />
m 3 2,2,7 GTP<br />
TΔS o<br />
ΔG o<br />
ΔH o<br />
280 290 300 310 320 330<br />
Temperature (K)
4.4.7. Biological Implications <strong>of</strong> Non-Linear van't H<strong>of</strong>f <strong>Thermodynamic</strong>s<br />
<strong>Thermodynamic</strong> features <strong>of</strong> the process <strong>of</strong> recognition <strong>of</strong> the 5' cap structure by<br />
<strong>eIF4E</strong> can provide an <strong>eIF4E</strong>-dependent switch for several in vivo metabolic pathways, as<br />
follows:<br />
1. The temperature-invariability <strong>of</strong> ΔG° for the natural dinucleotide cap analogues is <strong>of</strong><br />
special biological importance, because the regulation <strong>of</strong> <strong>eIF4E</strong> and its inhibitory<br />
binding protein, 4E-BP1, is affected by heat shock which causes an increase <strong>of</strong> the<br />
association between <strong>eIF4E</strong> and 4E- BP1 81 . Increased binding <strong>of</strong> 4E-BP1 to <strong>eIF4E</strong><br />
interferes <strong>with</strong> the formation <strong>of</strong> the active eIF4F translation initiation complex 31<br />
during heat shock 41,254-256 . As <strong>mRNA</strong>s <strong>of</strong> heat shock proteins are relatively cap-<br />
independent 257,258 , the enhanced inhibition <strong>of</strong> <strong>eIF4E</strong> by 4E-BP1 together <strong>with</strong> the<br />
temperature-invariant cap-binding affinity <strong>of</strong> <strong>eIF4E</strong> could provide a mechanism for<br />
the selective up-regulation <strong>of</strong> the synthesis <strong>of</strong> heat shock proteins.<br />
2. The distinct thermodynamic parameters <strong>of</strong> m 7 GTP and m GTP<br />
7 , 2 , 2<br />
116<br />
3 can shift the<br />
biochemical equilibria related to splicing in eukaryotic cells during heat shock. Two<br />
types <strong>of</strong> capped RNAs exist in eukaryotes, <strong>mRNA</strong>s and small nuclear RNAs<br />
(snRNAs). Their 5' termini are methylated in the nucleus to yield m 7 GpppN<br />
structure. 259 After nuclear export to cytosol, the cap <strong>of</strong> snRNAs is further methylated<br />
the at the amino group <strong>of</strong> the 7-methylguanosine moiety, forming m GpppN<br />
7 , 2 , 2<br />
The trimethylguanosine cap is a fragment <strong>of</strong> a nuclear localization signal (NLS),<br />
which is responsible for import <strong>of</strong> the snRN-protein spliceosomal complexes<br />
(snRNPs) into the nucleus, 23,24 where they take part in pre-<strong>mRNA</strong> splicing. 25 The<br />
trimethylguanosine cap-dependent transport is enhanced by a m GpppN<br />
7 , 2 , 2 -specific<br />
nuclear import receptor, a protein called snurportin1, which binds the trimethyl-<br />
guanosine cap structure approximately three orders <strong>of</strong> magnitude more strongly than<br />
the monomethylguanosine cap. 260 Since the preference <strong>of</strong> <strong>eIF4E</strong> for m 7 GTP vs.<br />
GTP<br />
, 2 , 2<br />
3 is significantly diminuted at higher temperatures, snRNAs could be also<br />
m 7<br />
recruited to the translational machinery, thus contributing to inhibition <strong>of</strong> translation<br />
initiation. Moreover, the binding <strong>of</strong> <strong>eIF4E</strong> to the trimethylguanosine cap <strong>of</strong> snRNPs<br />
prevents recognition <strong>of</strong> this structure by snurportin1, hence the nuclear import and<br />
consequently splicing could be disturbed. This competition could also lead to<br />
significant inhibition <strong>of</strong> protein biosynthesis.<br />
3<br />
3<br />
. 22
3. In some primitive eukaryotes 29,261,262 and in some chordate species 28 the process <strong>of</strong><br />
trans-splicing causes that RNAs contain the trimethylguanosine cap structure. Even<br />
~ 70% <strong>of</strong> the <strong>mRNA</strong> population has the original cap replaced by 3 GpppN in the<br />
117<br />
m 7 , 2 , 2<br />
C. elegans nematode. Five <strong>eIF4E</strong>-like translation initiation factors were found in this<br />
organism, and three <strong>of</strong> them could bind both to m 7 GTP and m GTP<br />
7 , 2 , 2<br />
3<br />
. 55,263,264 The<br />
cellular role for the individual <strong>eIF4E</strong> is<strong>of</strong>orms is still not known, although some <strong>of</strong><br />
them are essential for viability <strong>of</strong> the worm embryos, as shown by RNA<br />
interference. 264<br />
4.4.8. Isothermal Enthalpy-Entropy Compensation in Congener Series<br />
The enthalpic and entropic contributions to the Gibbs free energies <strong>of</strong> binding <strong>of</strong><br />
<strong>eIF4E</strong> to the series <strong>of</strong> the cap analogues at 293.16 K (Table 4-8) appear to satisfy a linear<br />
relationship <strong>with</strong> the high correlation coefficient, R 2 = 0.975 (Fig. 4-35). The slope yields a<br />
compensation temperature, Tc = 399 ± 24 K. The result is exactly the same, irrespective <strong>of</strong><br />
the data set which is chosen for the dinucleotide cap analogues, i. e. either the couples <strong>of</strong><br />
o o the intrinsic ( Δ H 0 , S0<br />
Δ ) or observed (ΔH°, ΔS°) parameters. This means that although the<br />
resultant enthalpy and the entropy <strong>of</strong> interaction are significantly influenced by self-<br />
stacking, the general thermodynamic property <strong>of</strong> recognition <strong>of</strong> <strong>mRNA</strong> 5' cap by <strong>eIF4E</strong> is<br />
insensitive to it.<br />
Enthalpy-entropy compensation is a widely reported phenomenon, which is <strong>of</strong>ten<br />
called an extra-thermodynamic feature <strong>of</strong> complex molecular systems that fluctuate or<br />
interact <strong>with</strong> the aqueous medium. However, several authors made detailed and critical<br />
reviews <strong>of</strong> compensation in congener series, demonstrating that it is in majority a trivial<br />
observation or an artifact that follows immediately from applied experimental<br />
conditions. 199,265-267 There are three categories <strong>of</strong> isothermal enthalpy-entropy<br />
compensation that can be detected in a series <strong>of</strong> homologous compounds:
TΔΔS° (kJ mol -1 )<br />
40<br />
20<br />
Figure 4-35. Isothermal enthalpy-entropy compensation for the <strong>mRNA</strong> 5’ cap congener series at<br />
293 K. The enthalpy gain is always greater than the entropy loss that accompanies the association<br />
<strong>of</strong> more and more tightly binding cap analogues, irrespective <strong>of</strong> whether the binding is described by<br />
a zero () or non-zero () heat capacity change. The linear fitting was performed <strong>with</strong> weighting by<br />
errors <strong>of</strong> both TΔS° and ΔH° (Table 4-8). The errors are not shown for clarity.<br />
1. In case <strong>of</strong> the solvation thermodynamics <strong>of</strong> a solute series, or the binding<br />
thermodynamics <strong>of</strong> a ligand series, when the congeners differ in the number or size<br />
<strong>of</strong> similar substituents, then the linear compensation is a trivial manifestation <strong>of</strong> the<br />
presence <strong>of</strong> a single source <strong>of</strong> additivity. The series <strong>of</strong> the studied nine cap analogues<br />
contains several sources <strong>of</strong> additivity: phosphate groups at the ribose ring, methyl<br />
substituents at the 2-amino group <strong>of</strong> the 7-methylguanine moiety, different aromatic<br />
substituents for the 7-methyl group, and different second nucleosides. Hence, this<br />
case can be ruled out.<br />
2. If the range <strong>of</strong> binding free energy (ΔG°) that can be observed during experiments is<br />
small in relation to the binding enthalpy (ΔH°), due to some intrinsic features <strong>of</strong> an<br />
investigated system or due to experimental conditions, then the linear correlation is<br />
simply a consequence <strong>of</strong> the equation:<br />
ΔH° − TΔS° = ΔG° ≈ constant .<br />
0<br />
-20<br />
-40<br />
-60<br />
m 7 GTP<br />
bz 7 GTP<br />
p-Cl-bz 7 GTP<br />
m 7 GDP<br />
m 7 GpppG<br />
m 7 Gppppm 7 G<br />
-100 -80 -60 -40 -20 0<br />
The ΔG° values <strong>of</strong> the <strong>eIF4E</strong>-cap association are always greater than 50 % <strong>of</strong> the<br />
corresponding ΔH° values (Fig. 4-36), which allows to exclude this case, too.<br />
118<br />
2,2,7<br />
m3 GTP<br />
m 7 GMP<br />
m 7 GpppC<br />
ΔH° (kJ⋅mol -1 )
ΔG° /ΔH°<br />
2.0<br />
1.5<br />
1.0<br />
0.5<br />
0.0<br />
119<br />
Figure 4-36. Proportion <strong>of</strong> ΔG° to<br />
ΔH° for association <strong>of</strong> cap analogues<br />
to <strong>eIF4E</strong> at 293 K<br />
3. True extra-thermodynamic compensation that reflects some additional information<br />
about a system, e. g. about the distribution <strong>of</strong> energy levels available to the system or<br />
about interactions between components, can occur when the two above mentioned<br />
cases were eliminated. The only problem that remains to be resolved consists in an<br />
answer to the question whether enthalpy-entropy compensation does not result<br />
fortuitously from high correlation between errors <strong>of</strong> ΔH° and ΔS° obtained by the<br />
van't H<strong>of</strong>f method.<br />
-45 -40 -35 -30 -25<br />
ΔG° (kJ⋅mol -1 )<br />
A statistical test has been used to determine the significance <strong>of</strong> the observed ΔH° vs.<br />
ΔS° plot for the series <strong>of</strong> the cap analogues. 266,267 The most exact criterion is to check<br />
whether the experimental temperature (Texp) lies outside the 95 % confidence interval for<br />
the compensation temperature: Texp < Tc − 2⋅σ or Tc + 2⋅σ < Texp. The 95 % interval is 351<br />
– 447 K, while the harmonic mean value <strong>of</strong> the experimental temperatures applied for the<br />
van't H<strong>of</strong>f analysis is ~ 297 K, as calculated from Table 4-7. Hence, the difference between<br />
Tc and Texp is even more than 4⋅σ, which unambiguously points to strong, non-trivial,<br />
isothermal enthalpy-entropy compensation.<br />
4.4.8.1. Molecular Interpretation <strong>of</strong> Isothermal Enthalpy-Entropy Compensation<br />
It is <strong>of</strong>ten suggested that enthalpy-entropy compensation is an intrinsic property <strong>of</strong><br />
complex systems that have many s<strong>of</strong>t modes <strong>of</strong> fluctuations, e. g. aqueous solutions and<br />
soluble proteins. A statistical mechanical analysis <strong>of</strong> an entirely general model <strong>of</strong> a<br />
significantly perturbed complex system revealed that the compensation temperature<br />
depended on where the energy <strong>of</strong> the perturbed state (U') lay <strong>with</strong> respect to the mean
energy <strong>of</strong> the unperturbed system (E). 199 However, any clear interpretation for the Tc value<br />
was not proposed.<br />
It seems that Tc can be directly related to the difference in stability <strong>of</strong> the system in<br />
the unperturbed and perturbed states. In other words, the difference between the<br />
compensation temperature and the harmonic mean experimental temperature can be a<br />
measure <strong>of</strong> the fluctuations <strong>of</strong> the unperturbed system, which are modulated by the<br />
perturbation. According as Tc is greater or lower than Texp, the fluctuations are silenced or<br />
enhanced, so the perturbation acts either stabilizing or destabilising, respectively. Thus, the<br />
Tc value <strong>of</strong> 399 ± 24 K shows that the energy (U') <strong>of</strong> the state <strong>of</strong> <strong>eIF4E</strong>, which binds to the<br />
cap structure lies much below the mean energy <strong>of</strong> the apo-protein (E):<br />
E − U' = 9.66 ± 1.7 kJ/mol.<br />
This rather large value, far exceeding RT (~ 2.5 kJ/mol), and almost comparable <strong>with</strong> the<br />
stabilization energy <strong>of</strong> globular proteins <strong>of</strong> similar size and hydrophobicity (~ 50 kJ/mol), 11<br />
suggests that the apo-<strong>eIF4E</strong> is a highly fluctuating, unstable protein, and only the specific<br />
cap binding provides enough stiffening <strong>of</strong> the global protein structure to make it stable on<br />
an usual level. This conclusion is in very good accordance <strong>with</strong> the observations gathered<br />
on other fields:<br />
1. biochemical: the great instability index calculated from the <strong>eIF4E</strong> amino acid<br />
sequence; 44<br />
2. biophysical: extensive conformational changes <strong>of</strong> <strong>eIF4E</strong> upon cap binding; the<br />
significant decrease <strong>of</strong> the amount <strong>of</strong> the active protein even at medium temperature;<br />
aggregation;<br />
3. biological: much lower concentration <strong>of</strong> <strong>eIF4E</strong> than that <strong>of</strong> the other translation<br />
initiation factors; the presence <strong>of</strong> <strong>eIF4E</strong> bound to 4E-BP or to eIF4G <strong>with</strong>in the<br />
eIF4F complex in the living cell; regulation <strong>of</strong> <strong>eIF4E</strong> biological activity by its<br />
cellular accessibility. 42<br />
In summary, the thermodynamics <strong>of</strong> the <strong>eIF4E</strong> association <strong>with</strong> the cap structure<br />
elucidates basic properties <strong>of</strong> this unusual protein, which are key to its biological activity.<br />
120
4.4.9. Inquiries about Origins <strong>of</strong> the Unusual Positive Heat Capacity<br />
Change<br />
Usually observed large, negative values <strong>of</strong> standard heat capacity change are<br />
characteristic for specific hydrophobic binding between proteins and nucleic acids as well<br />
as for protein folding. 131,134,194,196,242,268-271 The main negative contribution to<br />
121<br />
o<br />
p<br />
Δ C comes<br />
from the hydrophobic effect, i.e. the removal <strong>of</strong> non-polar and aromatic molecular surface<br />
from water upon complex formation. 131 On the contrary, the reduction <strong>of</strong> the polar surface<br />
area gives positive contribution to<br />
o<br />
Δ Cp<br />
, although to less extent, 2.3-fold 134 or 1.7-<br />
fold 130,268 , depending on the proposed model. Other contributions to negative<br />
o<br />
ΔCp<br />
comprise changes in s<strong>of</strong>t internal vibrational modes and temperature-dependent<br />
conformational changes which lead to the overall decrease in mobility <strong>of</strong> residues and to<br />
stabilization <strong>of</strong> relative arrangement <strong>of</strong> domains, and/or protein aggregation 131 . For<br />
instance, a large<br />
o<br />
p<br />
Δ C value <strong>of</strong> −1.58 ± 0.06 kJ/mol⋅K for water mediated antigen –<br />
antibody association is completely unrelated to the hydrophobic effect. Instead, this results<br />
only from an interdomain induced fit <strong>of</strong> the heavy and light polypeptide chains, which was<br />
proved by resolving <strong>of</strong> the crystal structures <strong>of</strong> the antibody in the free and antigen-bound<br />
forms. 243<br />
A process <strong>of</strong> protein-ligand interaction characterized itself by<br />
o<br />
p<br />
Δ C = 0 can also give<br />
rise to a non-zero, positive or negative heat capacity change, due to thermodynamic<br />
coupling <strong>with</strong> other possible equilibrium transitions 122,272 : proton uptake or dissociation,<br />
binding <strong>of</strong> the second ligand, a conformational change <strong>of</strong> the protein and/or the ligand.<br />
While the positive<br />
o<br />
p<br />
Δ C relevant to the protein unfolding is a common observation,<br />
examples reported for intermolecular interactions are extremely rare and contain few data,<br />
e.g. the formation <strong>of</strong> the phosph<strong>of</strong>ructokinase tetramer 123 , the interaction <strong>of</strong> the brain<br />
natriuretic peptide <strong>with</strong> heparin 273 , cobalt hexamine and spermidine binding to DNA, 274<br />
anion-exchange adsorption <strong>of</strong> cytochrome b5 and its surface-charge mutants. 275 On the<br />
other hand, it was shown for the interaction <strong>of</strong> c-Myb DNA-binding domain (R2R3) <strong>with</strong><br />
its target DNA, that the heat capacity change can be strongly temperature- and ionic<br />
strength-dependent, leading even to the sign inversion <strong>of</strong><br />
o<br />
p<br />
Δ C <strong>with</strong>in some ranges <strong>of</strong> those<br />
parameters 276 . In each case, Coulombic interactions were involved into the binding<br />
processes.
4.4.9.1. Changes <strong>of</strong> Solvent Accessible Surfaces Upon Binding<br />
The kinetic studies <strong>of</strong> the <strong>eIF4E</strong> − cap interaction by means <strong>of</strong> stopped-flow<br />
fluorescence spectroscopy as well as Brownian molecular dynamics simulations 4 revealed<br />
two-step character <strong>of</strong> the complex formation. The first step is the diffusionally and<br />
electrostatically controlled encounter <strong>of</strong> the protein and the ligand and the second step is<br />
the internal rearrangement <strong>of</strong> the encounter complex. Detailed analysis <strong>of</strong> the salt effect on<br />
the equilibrium association constants revealed that an uptake <strong>of</strong> roughly 65 water<br />
molecules to the first-layer hydration shell is necessary for the complex formation (see<br />
4.2.3.1., p. 74). This large hydration effect is relevant to significant conformational change<br />
<strong>of</strong> the protein upon the second step <strong>of</strong> the cap binding (see 4.2.3.1.6., p. 82), and leads to<br />
an increase <strong>of</strong> the protein solvent accessible surface area. The cooperativity <strong>of</strong> cap-binding<br />
site <strong>with</strong> the eIF4G/4E-BP-binding site (see 4.3.2., p. 90) suggests that the hydrophobic<br />
dorsal surface recognized by the eIF4G and 4E-BP1 peptides becomes more accessible<br />
after the cap has been bound. To estimate the upper limit <strong>of</strong> the positive contribution to<br />
ΔCp° from hydration <strong>of</strong> the protein hydrophobic surface, one should subtract the number <strong>of</strong><br />
water molecules trapped inside the cap-binding centre (at least 16, as detected by<br />
crystallography 1 ) from the total number <strong>of</strong> waters taken up to the complex (ΔN ≈ 65), since<br />
water in internal cavities is usually stiffly bound. 226,227 Since the surface hydrated by one<br />
water molecule is ~ 9.5 Å 2 , 119,232 then, even if one assumes that all remaining 49 water<br />
molecules hydrate a purely aliphatic surface <strong>of</strong> ~ 465 Å 2 (which is unlikely), and if one<br />
takes the maximal increment <strong>of</strong> ΔCp° per 1 Å <strong>of</strong> aliphatic surface (+2.14 J/mol⋅K 130 ), the<br />
o<br />
p<br />
calculated contribution to Δ C would equal only about +1 kJ/mol⋅K.<br />
Some additional contribution to the positive<br />
122<br />
o<br />
p<br />
Δ C arises from the burial <strong>of</strong> the<br />
uncharged and charged polar groups in the protein binding centre. Heat capacity changes<br />
arising from dehydration <strong>of</strong> nucleic acid components were calculated recently on the basis<br />
<strong>of</strong> polar and apolar accessible surface areas as +52.63 J/mol⋅K for guanine, −36.07 J/mol⋅K<br />
for ribose, and +46.02 J/mol⋅K for a single phosphate group. 277 Neglecting neighbour<br />
effects between base, sugar, and phosphate groups in the entire cap analogue, and the<br />
unknown resultant influence <strong>of</strong> the guanosine methylation which provides both the<br />
additional aliphatic group and the positive charge to the ring, the estimated range <strong>of</strong><br />
contribution from burial <strong>of</strong> 7-methylguanosine triphosphate would be about +0.2 kJ/mol⋅K.
4.4.9.2. Electrostatic Contributions to Heat Capacity Changes<br />
Careful inspection <strong>of</strong> the crystallographic 1,48,75 and NMR 76 structural data shows that<br />
stabilization <strong>of</strong> the <strong>mRNA</strong> 5' cap – <strong>eIF4E</strong> complexes is accomplished to great extent by<br />
charge-related interactions and partially complemented by the van der Waals contacts (see<br />
4.2., p. 60). Many charged groups are removed from water upon the association. The<br />
negatively charged 5'-5' phosphate chain <strong>of</strong> 7-methylGpppG is a primary anchor to the<br />
positively charged Arg112, Lys162, Arg157 and Lys159 side-chains <strong>of</strong> <strong>eIF4E</strong>. This<br />
charge-to-charge anchoring makes it possible to form further specific polar contacts in the<br />
narrow binding slot: cation-π sandwich stacking <strong>of</strong> the 7-methylguanine moiety <strong>with</strong><br />
Trp56 and Trp102, and three hydrogen bonds <strong>with</strong> Glu103 and Trp102.<br />
Hydrophobic area-based models <strong>of</strong> the heat capacity changes take into account only<br />
short-rang effects, while there are direct electrostatic contributions to heat capacity, which<br />
do not scale <strong>with</strong> the non-polar and polar surface areas. As shown by the finite-difference<br />
Poisson-Boltzmann method, three components <strong>of</strong> the total electrostatic contribution to<br />
o<br />
p<br />
Δ C are related to: (1) the rearrangement <strong>of</strong> water dipoles upon binding, (2) the<br />
redistribution <strong>of</strong> mobile ions in the solvent upon binding, and (3) the coupling between the<br />
dipolar and ionic terms. 278 The overall contribution <strong>of</strong> electrostatic interactions to<br />
123<br />
o<br />
p<br />
Δ C due<br />
to dehydration <strong>of</strong> charged residues upon protein – ligand binding is dominated by the<br />
positive term arising from the dielectric behaviour <strong>of</strong> water, which is opposed by the<br />
contribution <strong>of</strong> mobile solvent ions. Therefore, in addition to burial <strong>of</strong> polar or polarizable<br />
surface, changes in the water structure that accompany dehydration <strong>of</strong> ionized groups can<br />
also partially account for the observed positive<br />
o<br />
p<br />
Δ C . However, the possible electrostatic<br />
contribution from the phosphate groups and the basic amino acid side-chains is still at least<br />
one order <strong>of</strong> magnitude too small to explain the huge positive heat capacity changes for the<br />
cap analogues.<br />
The cation - π stacking itself has also a great electrostatic component, resulting from<br />
the attraction between the cation and the quadrupole charge distribution <strong>of</strong> the aromatic<br />
ring, which dominates the polarizability and dispersive forces. 129,148,149,279,280 In light <strong>of</strong> the<br />
above mentioned possible explanation <strong>of</strong> the positive<br />
seemed to be very interesting to check whether the unusual values <strong>of</strong><br />
o<br />
p<br />
Δ C by Coulombic contributions, it<br />
stacking <strong>of</strong> tryptophan <strong>with</strong> the non-typical, large, 7-methylguanosine cation.<br />
o<br />
p<br />
Δ C could rise from
4.4.9.3. Tryptophan Stacking <strong>with</strong> Cationic 7-Methylguanosine Moiety<br />
In order to extract information concerning the thermodynamics <strong>of</strong> the cationic 7-<br />
methylguanosine moiety interactions, NMR spectroscopy has been applied to examine two<br />
model systems: 7-methylGMP and tryptophan N-acetylamid, and a dinucleotide cap<br />
analogue, 7-methylGpppG, interacting <strong>with</strong> a dodecapeptide <strong>of</strong> the sequence related to a<br />
part <strong>of</strong> the <strong>eIF4E</strong> cap-binding site around Trp102 (DGIEPMWEDEKN). Stacking between<br />
7-methylG and tryptophan shifts the 1 H signals upfield, yielding microscopic equilibrium<br />
association constants (K), listed for the tryptophan protons at 298 K in Table 4-14,<br />
according to the titration curves shown in Fig. 4-37.<br />
Similar microscopic association constants for the individual tryptophan protons,<br />
related to stacking <strong>with</strong> m 7 GMP suggest that configuration <strong>of</strong> the two heteroaromatic rings<br />
is almost parallel. The dodecapeptide tryptophan interacts only <strong>with</strong> the methylated base <strong>of</strong><br />
m 7 GpppG, and the complex assumes both parallel and perpendicular orientations <strong>of</strong> the 7-<br />
methylguanosine moiety in relation to the indol ring in a dynamic equilibrium. 7,281 The<br />
presence <strong>of</strong> multiple forms <strong>of</strong> the complexes causes that the chemical environment <strong>of</strong> the<br />
tryptophan protons is affected in different manner, which yields the microscopic K for<br />
Table 4-14. Microscopic equilibrium association constants for tryptophan protons, related to<br />
stacking <strong>of</strong> tryptophan N-acetylamid <strong>with</strong> m 7 GMP at pH 5.6, and to binding <strong>of</strong> tryptophancontaining<br />
dodecapeptide <strong>with</strong> m 7 GpppG at pH 5.2; temperature 298 K.<br />
Tryptophan protons: H(2) H(4) H(5) H(6) H(7)<br />
Model system K (M -1 )<br />
Trp(N-aa)-m 7 GMP 15.9 ± 3.8 6.5 ± 1.6 6.7 ± 2.0 5.9 ± 1.8 7.8 ± 1.3<br />
Trp(pept)-m 7 GpppG 242 ± 67 61.8 ± 7.5 51.4 ± 7.2 39.4 ± 9.5 n. d.<br />
a not determined because <strong>of</strong> too weak changes <strong>of</strong> H(7) chemical shift <strong>with</strong> m 7 GpppG concentration<br />
Δδ HTrp (ppm)<br />
0.00<br />
-0.02<br />
-0.04<br />
-0.06<br />
-0.08<br />
H(2)<br />
H(6)<br />
H(5)<br />
H(4)<br />
H(7)<br />
0 5 10 15 20 25 30<br />
m 7 GMP (mM)<br />
Figure 4-37. Differences <strong>of</strong> 1 H chemical shifts <strong>of</strong> tryptophan N-acetylamid at 1 mM due to<br />
stacking upon titration <strong>with</strong> m 7 GMP at pH 5.6 (left); and <strong>of</strong> the dodecapeptide tryptophan at 2.8<br />
mM due to stacking upon titration <strong>with</strong> m 7 GpppG at pH 5.2 (right) at 298 K.<br />
Δδ HTrp (ppm)<br />
124<br />
0.01<br />
0.00<br />
-0.01<br />
-0.02<br />
-0.03<br />
-0.04<br />
0 3 6 9 12 15<br />
m 7 GpppG (mM)
Δδ HTrp (ppm)<br />
Figure 4-38. Temperature dependence <strong>of</strong> differences <strong>of</strong> 1 H chemical shifts <strong>of</strong> tryptophan Nacetylamid<br />
at 1 mM in the presence <strong>of</strong> 29.8 mM m 7 GMP at pH 5.6 (left); and <strong>of</strong> the dodecapeptide<br />
tryptophan at 2.8 mM in the presence <strong>of</strong> 17.2 mM m 7 GpppG at pH 5.2 (right).<br />
H(2) about 5-fold greater than K for the remaining protons, except H(7). The H(7) proton<br />
<strong>of</strong> the dodecapeptide tryptophan exercises a slight downfield shift.<br />
Although stacking is thought <strong>of</strong> as a hydrophobic interaction, binding <strong>of</strong> tryptophan<br />
<strong>with</strong> both cationic 7-methylGMP and 7-methylGpppG is enthalpy-driven and entropy-<br />
opposed, <strong>with</strong>out significant heat capacity changes in the temperature range <strong>of</strong> 278–320 K,<br />
as determined on the basis <strong>of</strong> the temperature-dependent differences <strong>of</strong> 1 H chemical shifts<br />
<strong>of</strong> the tryptophan protons (Fig. 4-38, Table 4-15). Satisfactory goodness <strong>of</strong> fit in terms <strong>of</strong><br />
the sum-<strong>of</strong>-squares <strong>of</strong> deviations (R 2 ) has been obtained when it is assumed that the<br />
stacking is described by the constant values <strong>of</strong> the van't H<strong>of</strong>f enthalpy change (ΔH°vH) and<br />
the entropy change (ΔS°). However, the P-value that refers to the probability <strong>of</strong> random<br />
distribution <strong>of</strong> fitting residuals along the fitted curve is slightly improved if a non-zero<br />
o<br />
Δ Cp<br />
value is allowed for the m 7 GMP-Trp interaction (Fig. 4-39). The heat capacity change<br />
estimated in this way is positive but small, from +0.12 to +0.18 kJ/mol⋅K for different<br />
tryptophan protons, charged by the relative numerical error <strong>of</strong> ~ 100 %. It seems unlikely<br />
that the 7-methylguanosine sandwich stacking between Trp102 and Trp56 <strong>with</strong>in the cap-<br />
binding site <strong>of</strong> <strong>eIF4E</strong> could alone provide a positive contribution to<br />
o<br />
p<br />
explain the observed Δ C values <strong>of</strong> the order <strong>of</strong> +5 kJ/mol⋅K.<br />
R 2 , P value<br />
-0.02<br />
-0.06<br />
-0.10<br />
-0.14<br />
1.0<br />
0.5<br />
0.0<br />
280 290 300 310 320<br />
Temperature (K)<br />
-1000 -500 0 500 1000<br />
ΔC p° (J/mol⋅K)<br />
H(2)<br />
H(6)<br />
H(5)<br />
H(4)<br />
H(7)<br />
125<br />
Δδ HTrp (ppm)<br />
-0.02<br />
-0.04<br />
-0.06<br />
-0.08<br />
280 290 300 310<br />
Temperature (K)<br />
o<br />
p<br />
H(2)<br />
H(7)<br />
H(6)<br />
H(5)<br />
H(4)<br />
Δ C great enough to<br />
Figure 4-39. Goodness <strong>of</strong> fit (R2, ) and<br />
probability <strong>of</strong> random distribution <strong>of</strong><br />
fitting residuals (P value, ) for curves<br />
fitted to temperature dependence <strong>of</strong><br />
ΔδH(7) <strong>of</strong> tryptophan N-acetylamid<br />
stacking <strong>with</strong> m 7 GMP at pH 5.6 <strong>with</strong> fixed<br />
ΔCp° values indicated in figure.
Table 4-15. <strong>Thermodynamic</strong> parameters for stacking <strong>of</strong> <strong>mRNA</strong> 5' cap analogues <strong>with</strong> tryptophan<br />
N-acetylamid and the tryptophan-containing dodecapeptide. The van't H<strong>of</strong>f enthalpy change<br />
(ΔH°vH) and the entropy change (ΔS°) are approximately constant <strong>with</strong> temperature. For reference<br />
the parameters <strong>of</strong> <strong>eIF4E</strong>-m 7 GpppG interaction at 293 K: ΔH°vH = −65 ± 31 kJ/mol, ΔS° = −91 ± 58<br />
J/mol⋅K (Table 4-8)<br />
Tryptophan protons: H(2) H(4) H(5) H(6) H(7)<br />
Model system<br />
Trp(N-aa)-m<br />
ΔH°vH (kJ/mol)<br />
7 GMP<br />
Trp(pept)-m<br />
-26.6 ± 1.8 -25.87 ± 0.78 -26.3 ± 1.4 -25.7 ± 1.1 -26.44 ± 0.79<br />
7 GpppG n. d. a<br />
-29.6 ± 3.2 -33.8 ± 4.1 -31.8 ± 4.6 -35.1 ± 4.1<br />
ΔS° (J/mol⋅K)<br />
Trp(N-aa)-m 7 GMP -65.8 ± 4.6 -64.0 ± 1.9 -65.1 ± 3.4 -62.4 ± 2.7 -64.1 ± 2.0<br />
Trp(pept)-m 7 GpppG n. d. a<br />
-71.3 ± 8.6 -81 ± 12 -75 ± 13 -89 ± 12<br />
a<br />
not determined due to H(2) and H(6) signal overlapping at higher temperatures<br />
The ΔH°vH values for stacking <strong>of</strong> the 7-methylG moiety <strong>with</strong> tryptophan are ~2-fold<br />
greater than those reported for adenine and uracil base stacking (−12.6 ÷ −14.2 kJ/mol per<br />
stack), 252,282,283 and for intramolecular self-stacking <strong>of</strong> the dinucleotide cap analogues 198<br />
(1ΔH°, Table 4-11). The enthalpy and entropy changes <strong>of</strong> the m 7 G-Trp stacking seem to<br />
provide a significant contribution to the overall thermodynamic parameters <strong>of</strong> cap binding<br />
to <strong>eIF4E</strong> upon translation initiation. The 7-methylG moiety represents a unique example <strong>of</strong><br />
a cation which is concurrently a heteroaromatic ring. The large, negative values <strong>of</strong> both<br />
ΔH°vH and ΔS° for stacking <strong>of</strong> 7-methylG <strong>with</strong> the protein tryptophan or <strong>with</strong> the second<br />
base <strong>with</strong>in the dinucleotide cap should be attributed to the dominating Coulombic<br />
character <strong>of</strong> the cation - π interactions. 129,148,149,279,280<br />
Figure 4-40. Sandwich stacking <strong>of</strong> 7-methylG moiety in between two absolutely conserved<br />
tryptophans in the <strong>mRNA</strong> 5’ cap-binding centres <strong>of</strong> murine 75 (left) and yeast 76 (right) translation<br />
initiation factor <strong>eIF4E</strong>. The structure <strong>of</strong> the human complex 48 is identical to the murine one.<br />
126
The results obtained for the model systems point to a diversity <strong>of</strong> permissible spatial<br />
structures <strong>of</strong> the stacked complexes. Similar diversity is observed among the sandwich<br />
configurations in the <strong>eIF4E</strong> cap-binding site. The structures are becoming more parallel in<br />
the course <strong>of</strong> evolution, from primitive eukaryotes like yeast 76 to higher ones, like mouse 75<br />
or human 48 (Fig. 4-40).<br />
4.4.9.4. Coupling <strong>of</strong> Other Intermolecular Equilibria to <strong>eIF4E</strong> – Cap Binding<br />
The possible origin <strong>of</strong> the positive heat capacity changes can be also connected to<br />
coupled equilibrium transitions. If coupled equilibria are occurring and/or significant<br />
concentrations <strong>of</strong> intermediate complexes are present at equilibrium, then measured<br />
association constants may depend on the total concentration <strong>of</strong> protein and/or ligand. 118,284<br />
The question arises why the Kas values for <strong>eIF4E</strong> – cap interactions do not depend on the<br />
concentration range (see 4.1.2., p. 51).<br />
The processes that are coupled to the <strong>eIF4E</strong> – cap binding take place <strong>with</strong><br />
participation <strong>of</strong> environment components. The partial protonation is immediately<br />
equilibrated by buffer ionization (Hepes at 50 mM), and the molar concentration <strong>of</strong> water<br />
(~ 50 M) and potassium cations (0.1 M) is much greater than those <strong>of</strong> the protein and the<br />
ligand (nM to μM range). Self-stacking <strong>of</strong> the dinucleotide cap analogues is weak and the<br />
conformational change is fast.<br />
The intermediate complex life-time can be calculated from the kinetic data obtained<br />
by the stopped-flow method. 4 The encounter rate constant for the association <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong><br />
m 7 GpppG was k+1 = 1.59 ± 0.07 ⋅ 10 8 M -1 ⋅s -1 (at 150 mM KCl), the rate for dissociation <strong>of</strong><br />
the encounter complex was k−1 = 50 ± 8 s -1 , the rate for internal rearrangement to the final<br />
conformation was k+2 = 99 s -1 , and the rate for the reverse process was k−2 = 6 s -1 . Hence,<br />
at typical protein concentration range that was applied for the Kas determination ([Pact]<br />
from 0.01 μM to 1 μM) and at the saturating concentration <strong>of</strong> the ligand, the equilibrium <strong>of</strong><br />
the second step is much more shifted toward the final product than the equilibrium <strong>of</strong> the<br />
first step that leads to the intermediate state:<br />
k + 2 k + 1 ⋅[<br />
Pact<br />
]<br />
>><br />
k k<br />
−2<br />
−1<br />
Additionally, the cap analogue concentration is far from the saturation state during the<br />
whole course <strong>of</strong> the titration, so the inequality is still stronger. Therefore, the only<br />
significant rate-limiting step during the overall association is the diffusionally and<br />
127
electrostatically driven first encounter <strong>of</strong> <strong>eIF4E</strong> and cap, and the observed Kas does not<br />
depend on the protein concentration.<br />
Having removed the doubts regarding the lack <strong>of</strong> influence <strong>of</strong> the coupled equilibria<br />
on the values <strong>of</strong> the association constants one can consider putative contributions to<br />
from the thermodynamic coupling.<br />
128<br />
o<br />
ΔCp<br />
1. Self stacking <strong>of</strong> dinucleotide cap analogues is mandatory coupled to <strong>eIF4E</strong><br />
association <strong>with</strong> cap, and yields a negligible negative contribution to the observed<br />
o<br />
p<br />
Δ C (see 4.4.4., p. 110).<br />
2. Partial protonation <strong>of</strong> the N(1) position <strong>of</strong> the cap analogue, the potassium cation<br />
release from the phosphate chain, and uptake <strong>of</strong> ~ 65 water that accompanies<br />
conformational changes <strong>of</strong> the protein upon cap binding (see 4.2.3.1., p. 74)<br />
represent the case <strong>of</strong> non-mandatory coupling, and hence can provide positive<br />
o<br />
p<br />
contributions to the overall Δ C .<br />
In summary, the following effects can together yield the positive<br />
several kJ/mol⋅K: 131<br />
• the hydration <strong>of</strong> the hydrophobic dorsal surface (up to ~ +1 kJ/mol⋅K),<br />
o<br />
p<br />
Δ C at the level <strong>of</strong><br />
• the dehydration <strong>of</strong> the ligand and protein ionized groups in the binding site (up to ~ 0.4<br />
kJ/mol⋅K for cation-π stacking, and up to ~ 0.5 kJ/mol⋅K for dehydration <strong>of</strong> the other<br />
charged and uncharged polar surfaces),<br />
• and the thermodynamic coupling <strong>with</strong> accompanying equilibrium transitions<br />
A kind <strong>of</strong> difficulty in interpretation is related to the lack <strong>of</strong> the apo-<strong>eIF4E</strong> structure,<br />
which is necessary for the complete analysis.<br />
4.4.10. Linear Correlation between ΔCp° and ΔG°<br />
The values <strong>of</strong><br />
o<br />
Δ C p appear to correlate linearly <strong>with</strong> the intrinsic free energy <strong>of</strong><br />
the binding (ΔG°0): the stronger the binding the less positive<br />
o<br />
Δ C p (Fig. 4-41). The<br />
apparent correlation can be fortuitous but it can also consist in any causality, as the<br />
correlation coefficient (r 2 ) equals 0.91. The cap analogues <strong>of</strong> the highest binding affinity,<br />
e.g. 7-methylGTP, do not reveal any observable curvature <strong>of</strong> the van't H<strong>of</strong>f plot.<br />
Systematic ΔC°p dependence on the cap affinity for <strong>eIF4E</strong> suggests that the common<br />
positive contribution can be compensated to different extent by the negative contribution
elated to the binding specificity. Most probably, this can result from tightening <strong>of</strong> the<br />
protein global fold upon binding, that yields the negative contribution to<br />
129<br />
o<br />
p<br />
Δ C , similarly as<br />
in the case <strong>of</strong> the specific antigen-antibody association. 131,243 This hypothesis is supported<br />
by the presence <strong>of</strong> the non-trivial enthalpy-entropy compensation discussed above. It was<br />
shown that the preferential ligand binding to the lower microstates <strong>of</strong> a protein led to a<br />
shift in the distribution <strong>of</strong> the microstates, and, consequently, reduced width <strong>of</strong> the<br />
distribution implied a decrease in heat capacity upon ligand binding. 122 Thus, for the most<br />
specific cap analogues the negative stabilization effect related to the specificity <strong>of</strong> the<br />
binding can make the<br />
experimental data. 248<br />
ΔCp°° (kJ/mol⋅K)<br />
6<br />
3<br />
0<br />
-3<br />
-6<br />
-9<br />
o<br />
p<br />
Δ C value too small to be discerned <strong>with</strong>in the noise <strong>of</strong> the<br />
-70 -60 -50 -40 -30 -20 -10 0 10<br />
ΔG° (kJ/mol)<br />
<strong>eIF4E</strong> + <strong>mRNA</strong> 5' cap analogues<br />
DNA + intercalators<br />
L-isoleucine tRNA ligase + substrates<br />
serine proteases + Na +<br />
protein + DNA (specific)<br />
protein + DNA (nonspecific)<br />
Figure 4-41. Correlation <strong>of</strong> heat capacity changes (ΔC°p) <strong>with</strong> standard molar binding free energies<br />
(ΔG°) for different intermolecular interactions.<br />
Linear ΔG° - ΔCp° correlation is rarely found, since it requires systematic studies on<br />
a consistent ligand series, while the suitable data obtained by time- and protein-consuming<br />
thermodynamic measurements are rather scattered. Several available examples are<br />
collected in Fig. 4-41. A general tendency is common but only two enthalpy driven<br />
processes involving charged ligands, i. e. specific sodium cation binding by serine<br />
proteases 285 and the <strong>eIF4E</strong> – cap binding yield remarkable slopes (0.93 ± 0.14 K -1 , and<br />
0.275 ± 0.044 K -1 , respectively). Binding <strong>of</strong> the L-isoleucine tRNA ligase to L-isoleucine<br />
and L-valine is not accompanied by any conformational changes <strong>of</strong> the large (120 kD),<br />
multidomain protein, which is known from the crystal structures <strong>of</strong> the native enzyme and
its complexes. 286 The heat capacity changes for binding <strong>of</strong> various substrates to the enzyme<br />
are approximately constant, though the range <strong>of</strong> the corresponding free energy changes<br />
encompasses almost 20 kJ/mol. 287 Drug - DNA intercalation is related partially to the<br />
binding-induced changes in non-polar and polar solvent-accessible surface areas, and<br />
consequently the binding free energy is proportional to the hydrophobic effect (slight<br />
dependence). 120,121,288 Specific interactions <strong>of</strong> DNA <strong>with</strong> repressors and RNA polymerase,<br />
driven by hydrophobicity <strong>of</strong> the specific sites, are characterized by the wide-spread protein<br />
conformational changes and large negative heat capacity changes, while non-specific<br />
interactions, driven only by the counterion release, are accomplished <strong>with</strong><br />
117,196,289-293<br />
130<br />
o<br />
Δ C p = 0. 112-<br />
However, no systematic literature data for the ΔG°-dependent positive heat capacity<br />
changes are available. Searching for origins <strong>of</strong> the positive<br />
o<br />
Δ C p and for the causal<br />
explanation <strong>of</strong> the apparent linear relationship in terms <strong>of</strong> statistical physics will be the<br />
subject <strong>of</strong> further investigations.<br />
4.4.11. Discussion <strong>with</strong> Other Authors<br />
The results described herein are contradictory to the first conclusions drawn from<br />
studies <strong>of</strong> 7-methylGTP and 7-methylGpppG binding to <strong>eIF4E</strong> from human<br />
erythrocytes 156 . Although the human 49 and murine 50 proteins are almost identical (98%<br />
homology for the full length proteins, and 100% for truncated (28-217) <strong>eIF4E</strong>), those<br />
results suggested that the <strong>eIF4E</strong> − cap binding was entropy-driven and enthalpy-opposed,<br />
<strong>with</strong> the constant thermodynamic parameters in the 278-308 K temperature range (ΔS° =<br />
+219 ± 11 J⋅mol -1 K -1 , ΔH° = +33.9 ± 1.7 kJ⋅mol -1 for 7-methylGpppG 156 ). However, the<br />
human protein was purified by means <strong>of</strong> the cap-affinity chromatography, what could<br />
result in up to 60% <strong>of</strong> the unremovable cap analogue bound to <strong>eIF4E</strong>. 1,208 This purification<br />
method yielded the apparent association constants up to 300-fold lower 5 then those<br />
measured for <strong>eIF4E</strong> purified <strong>with</strong>out contact <strong>with</strong> cap 1 (see Table 4-2, p. 61). Moreover,<br />
neither the decreasing temperature-dependence <strong>of</strong> the human <strong>eIF4E</strong> activity nor<br />
fluorescence <strong>of</strong> the cap analogues were taken into account. The authors also reported the<br />
fluorescence intensity <strong>of</strong> <strong>eIF4E</strong> and the analogues as invariant over the temperature range<br />
studied, which is impossible. Taken together, these main reasons could lead to<br />
misinterpretation <strong>of</strong> the experimental data.
The same reservation but even to much greater extent concerns the work <strong>of</strong> Shen et<br />
al. 294 Apparently higher association constants for dinucleotides than that for 7-methylGTP<br />
results simply from the inner filter effect, as no corrections were applied in spite <strong>of</strong> the<br />
huge concentration <strong>of</strong> <strong>eIF4E</strong> (10 μM) and the cap (22 μM). <strong>Thermodynamic</strong> parameters<br />
derived in such manner are fortuitous and do not allow a reasonable analysis.<br />
Recently, the human <strong>eIF4E</strong> − 7-methylGpppG binding affinity was fluorometrically<br />
studied in the context <strong>of</strong> cell growth suppression, and the association constant <strong>of</strong> 0.83 ±<br />
0.14 μM -1 was obtained <strong>with</strong> use <strong>of</strong> some corrections (at 296.2 K, pH 7.5, 300 mM<br />
KCl). 295 The corresponding Kas value reported herein for truncated murine <strong>eIF4E</strong> (28-217)<br />
is 5.13 ± 0.81 μM -1 (at 297.6 K, pH 7.2, 100 mM KCl, Table 4-7, p. 101). These results are<br />
in an excellent agreement, since elevation <strong>of</strong> KCl concentration from 100 to 300 mM<br />
causes a significant, ~ 6-fold decrease <strong>of</strong> the association constant due to screening <strong>of</strong> the<br />
electrostatic attraction between the basic amino acids in the cap-binding site <strong>of</strong> <strong>eIF4E</strong> and<br />
the phosphate chain <strong>of</strong> 7-methylGpppG (see 4.2.3.1., p. 74).<br />
4.4.12. Conclusions<br />
The positive heat capacity change at constant pressure appears to be a characteristic<br />
feature <strong>of</strong> <strong>eIF4E</strong> binding to <strong>mRNA</strong> 5' cap. The chemical cap analogues <strong>of</strong> the highest<br />
specificity exhibit the heat capacity change proportionally shifted toward less positive or<br />
undetectable values. Data obtained from two independent methods, fluorescence<br />
quenching and isothermal titration calorimetry provided excellently accordant<br />
thermodynamic parameters. The microcalorimetric results additionally yielded a<br />
quantitative confirmation <strong>of</strong> partial protonation <strong>of</strong> the ligand, which is necessary to form<br />
one <strong>of</strong> the crucial hydrogen bonds in the protein binding centre.<br />
Isothermal enthalpy-entropy compensation among the cap analogues <strong>of</strong> different<br />
specificity for <strong>eIF4E</strong> at 293 K points to several thermodynamic features: the dominating<br />
enthalpic character for the whole congener series, a great instability <strong>of</strong> the apo-form <strong>of</strong><br />
<strong>eIF4E</strong>, and to conformational rearrangement <strong>of</strong> the whole protein upon the complex<br />
formation, leading to the final, stable, cap-bound state.<br />
The exceptionally large positive<br />
131<br />
o Δ Cp<br />
<strong>of</strong> the binding can be attributed to<br />
simultaneous interplay <strong>of</strong> various intermolecular processes: the extensive additional<br />
hydration <strong>of</strong> the <strong>eIF4E</strong> hydrophobic dorsal surface, dehydration <strong>of</strong> the ionized groups,
including the 7-methylguanosine cation, and the burial <strong>of</strong> polar groups <strong>of</strong> the interacting<br />
molecules, as well as to the thermodynamically coupled differential ligand protonation and<br />
the potassium cation release from the ligand. The negative contribution cancelling the<br />
positive one for the most tightly binding analogues can arise from the global protein<br />
conformational change that stiffen the complex structure. The induced shift in the self-<br />
stacking equilibrium <strong>of</strong> the dinucleotide cap analogues gives a negligible negative<br />
o<br />
p<br />
contribution to the overall Δ C .<br />
Because <strong>of</strong> the non-zero heat capacity changes, the interactions <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> natural<br />
dinucleotide cap analogues are both enthalpy- and entropy-driven over the range <strong>of</strong><br />
biological temperatures, and stability <strong>of</strong> the <strong>eIF4E</strong> − cap complex is almost temperature-<br />
independent.<br />
The general description <strong>of</strong> the cap – <strong>eIF4E</strong> association is intricate due to strict<br />
synergy <strong>of</strong> charge-related and hydrophobic interactions <strong>with</strong>in the binding site and on the<br />
solvent accessible molecular surface <strong>of</strong> the complex. These properties make this molecular<br />
system unique in the thermodynamic sense.<br />
132
5. Summary<br />
The thesis was aimed at revealing the molecular mechanism <strong>of</strong> recognition <strong>of</strong> <strong>mRNA</strong><br />
5' cap structure by <strong>eIF4E</strong>, previously limited to a static view from the crystal structures.<br />
Chemically modified cap-analogues proved to be a valuable tool to probe contribution <strong>of</strong><br />
various types <strong>of</strong> molecular interaction to the <strong>eIF4E</strong>-cap complex stability via emission<br />
spectroscopy measurements. A fast and accurate method <strong>of</strong> synchronized fluorescence<br />
titration has been designed to obtain reliable equilibrium association constant values,<br />
which are not only relative to one another, but for the first time have an absolute meaning,<br />
and thus can be interpreted in terms <strong>of</strong> the free energy <strong>of</strong> binding. The mechanism <strong>of</strong> the<br />
complex formation at physiological pH can be now presented as an interplay <strong>of</strong> several<br />
intermolecular processes (Scheme 5-1). Recognition <strong>of</strong> the 5' cap structure by <strong>eIF4E</strong><br />
binding site begins <strong>with</strong> electrostatic attraction <strong>of</strong> the cap phosphate chain, from which one<br />
potassium cation is being removed. In the initial encounter complex ([<strong>eIF4E</strong>•cap]*) the<br />
cap is first anchored by the phosphate chain and next, cooperative cation - π sandwich<br />
stacking together <strong>with</strong> hydrogen bonding <strong>of</strong> 7-methylguanine occurs. Differential<br />
protonation at the N(1) position <strong>of</strong> 7-methylguanine is necessary to enhance stacking,<br />
which in turn conditions formation <strong>of</strong> three hydrogen bonds. The second step <strong>of</strong><br />
association is accompanied by preferential hydration involving ~ 65 water molecules.<br />
Some <strong>of</strong> them are trapped inside the cap-binding slot, while several tens supply the first-<br />
layer hydration shell <strong>of</strong> the complex. This extensive water reorganization implies a<br />
significant conformational change <strong>of</strong> the whole protein. The latter conclusion comes<br />
independently <strong>of</strong> several other sets <strong>of</strong> experimental results: isothermal enthalpy-entropy<br />
compensation, deaggregation <strong>of</strong> <strong>eIF4E</strong> forced by association <strong>with</strong> cap, cooperativity <strong>of</strong><br />
cap-binding and eIF4G/4E-BP1-binding sites.<br />
- 1⋅ K + + 0.5 ⋅ H +<br />
<strong>eIF4E</strong> + cap [<strong>eIF4E</strong> cap]* <strong>eIF4E</strong> cap<br />
133<br />
+ 65 ⋅ H2O<br />
Scheme 5-1. Proposed model <strong>of</strong> two-step association <strong>of</strong> <strong>eIF4E</strong> <strong>with</strong> the <strong>mRNA</strong> 5' cap
The appropriate equilibrium equation for <strong>eIF4E</strong>-cap binding has the following form:<br />
K<br />
t<br />
[ <strong>eIF4E</strong><br />
• cap][<br />
K ]<br />
=<br />
65 +<br />
[ <strong>eIF4E</strong>]<br />
[ cap]<br />
[ H O]<br />
[ H ]<br />
0<br />
0<br />
2<br />
+<br />
0.<br />
5<br />
(<strong>with</strong>out terms for buffer; index "0" denotes equilibrium concentrations <strong>of</strong> free species).<br />
Comparison <strong>of</strong> the <strong>eIF4E</strong>-binding affinity <strong>of</strong> natural vs structurally modified cap<br />
analogues together <strong>with</strong> the structures <strong>of</strong> the <strong>eIF4E</strong>-cap complexes makes a foundation to<br />
rational design <strong>of</strong> new cap analogues <strong>with</strong> better inhibitory properties than those<br />
synthesized hitherto. Quantitative analysis <strong>of</strong> the binding free energy shows that the cap<br />
triphosphate chain contributes in one half to it. Since the negative charge at the cap<br />
phosphate chain is so crucial for the efficient binding to <strong>eIF4E</strong>, it should be indispensably<br />
included in the designed agents. Hence, thinking about inhibition <strong>of</strong> the <strong>eIF4E</strong> cellular<br />
activity by 5' cap analogues must involve an active transport <strong>of</strong> the charged species through<br />
the cellular bilayer phospholipid membrane. One hopeful analogue is the p-Ch-bz7GTP,<br />
which strongly binds to <strong>eIF4E</strong> <strong>with</strong> the affinity <strong>of</strong> 10 7 – 10 8 M -1 . The equilibrium binding<br />
constant is relatively weakly temperature-dependent, since the association is both enthalpy-<br />
and entropy-driven. This artificial cap analogue should not be easily metabolized or<br />
trapped by other proteins, and thus it can be expected that its half lifetime could be long<br />
enough to render significant translation inhibition. This hope is supported by the<br />
observation that the overall inhibition constant (KI) <strong>of</strong> p-Ch-bz7GTP determined from<br />
biological in vitro assay in a rabbit reticulocyte lizate 68 is shifted toward lower values in<br />
comparison <strong>with</strong> that resulting from the average correlation for all cap analogues (Fig. 4-<br />
11, p. 63).<br />
The thermodynamic studies revealed unique, large and positive heat capacity<br />
changes that accompany the <strong>eIF4E</strong> – cap interactions. The binding <strong>of</strong> natural dinucleotide<br />
cap analogues are both enthalpy- and entropy-driven over the range <strong>of</strong> biological<br />
temperatures, and stability <strong>of</strong> the <strong>eIF4E</strong> − cap complex is almost temperature-independent.<br />
These findings are suitable for further development <strong>of</strong> quantitative interpretation <strong>of</strong><br />
intermolecular recognition specificity, and are also a kind <strong>of</strong> challenge for theoretical<br />
interpretation.<br />
The author hopes that the presented thesis contribute to more pr<strong>of</strong>ound insights into<br />
how <strong>eIF4E</strong> interacts <strong>with</strong> other components <strong>of</strong> the cytoplasmic machinery responsible for<br />
cap-dependent translation initiation.<br />
134
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