1784 F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 Fig. 7. Fluorescence emission quenching <strong>of</strong> Tyr15 <strong>of</strong> peptide H4 by FRET to SB 242784 (●). Curve corresponds to the theoretical expectation for FRET in the absence <strong>of</strong> inhibitor binding to peptide H4 (see text). The range <strong>of</strong> SB 242784 to total phospholipid ratios in these experiments was 0.18% to 1.8%. Inset: overlap between tyrosine fluorescence emission ( ) and SB242784 absorption spectrum (–––), R 0 (Tyr-SB 242784)=24 Å. When the FRET binding assay was applied to the peptide H4/SB 242784 system, the results were substantially different (Fig. 7). With this inhibitor, the obtained FRET efficiencies matched the theoretical expectations for a random distribution <strong>of</strong> acceptors, suggesting absence <strong>of</strong> binding between SB 242784 and the peptide. No uncertainty exists regarding this result as the position <strong>of</strong> the inhibitor inside the bilayer is accurately know from parallax fluorescence quenching techniques (12.8 Å from the center <strong>of</strong> the bilayer — [36]). 5. Discussion 5.1. Use <strong>of</strong> peptide H4 as a model peptide for the 4th trans<strong>membrane</strong> segment <strong>of</strong> subunit c Aggregation <strong>of</strong> trans<strong>membrane</strong> <strong>peptides</strong> containing a hydrophobic sequence and flanked by basic residues has already been reported [38–40], thus detection <strong>of</strong> aggregates <strong>of</strong> peptide H4 at low L/P ratios (
F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 1785 [26]. In the same way, c-subunit residues shown to be important for bafilomycin A 1 inhibitory activity through mutational <strong>studies</strong> [9] could not be directly involved in the binding <strong>of</strong> the inhibitor, but still play a role in the inhibition mechanism. Here, we proposed to further define the structural requirements for bafilomycin A 1 and SB 242784 binding to the V-ATPase, by focusing on the protein domain expected to be the primary contributor to the inhibitor binding site, the putative 4th trans<strong>membrane</strong> domain <strong>of</strong> the c-subunit. The extent <strong>of</strong> binding efficiencies achieved using only <strong>peptides</strong> corresponding to this section <strong>of</strong> the protein is an indicator <strong>of</strong> the relevance <strong>of</strong> the remaining protein domains to the binding <strong>of</strong> inhibitor, the first step in the inhibitory mechanism. No evidence was found supporting SB 242784 association <strong>with</strong> the peptide H4. On the other hand, bafilomycin A 1 was shown to possess only moderate affinity for the trans<strong>membrane</strong> <strong>peptides</strong>. Due to uncertainties in bafilomycin A 1 position in bilayers it was impossible to rigorously quantify a binding constant for the process, but a reliable range for this parameter could be presented. The differences in bafilomycin A 1 binding efficiencies for the peptide H4, H4c′ and H4 A ← E are not significant as they fall <strong>with</strong>in, or close to the error <strong>of</strong> the fits. For H4c′ this result is somewhat surprising. Mutations on conserved residues <strong>of</strong> the c-subunit known to confer resistance to bafilomycin A 1 and concanamycin were shown to be ineffective in the c′ and c′ is<strong>of</strong>orms [9]. It was proposed that if a bafilomycin A 1 binding site was present on these is<strong>of</strong>orms, then it must possess lower affinity than in the c is<strong>of</strong>orm [9]. If this is the case, then the differences in binding efficiencies between the two is<strong>of</strong>orms must be explained by discrepancies in the interaction <strong>of</strong> the inhibitor <strong>with</strong> parts <strong>of</strong> the protein other than the highly conserved putative 4th trans<strong>membrane</strong> helix. Although the contribution from the 4th trans<strong>membrane</strong> segment (TM4) to the binding site affinity for bafilomycin A 1 is not negligible, it is absolutely unable to explain the extremely low IC 50 <strong>of</strong> bafilomycin A 1 (0.1 nM). The binding efficiencies observed for concanamycin (IC 50 close to bafilomycin A 1 ) and the intact isolated c-subunit [27] were more than 1000 times larger than the ones recovered in the present study. Therefore, it is clear that either: (i) the other trans<strong>membrane</strong> segments from the c-subunit, namely TM1 and TM2, are essential in the formation <strong>of</strong> an efficient inhibitor binding site, or that (ii) interactions <strong>of</strong> TM4 <strong>with</strong> other protein segments are required for this protein section to assume the appropriate folding which enables the establishment <strong>of</strong> the necessary interactions for an efficient binding between the residues <strong>of</strong> the 4th trans<strong>membrane</strong> segment <strong>of</strong> the c-subunit and the inhibitors. Absence <strong>of</strong> SB 242784 binding to the peptide H4 could be explained by the much lower IC 50 <strong>of</strong> this inhibitor (26 nM in chicken osteoclasts [22]) that reflects a much lower binding site affinity for this inhibitor, and the absence <strong>of</strong> remaining c-subunit trans<strong>membrane</strong> segments potentiates this effect. In this work, in addition to the detailed comparison <strong>of</strong> the different inhibitors and <strong>peptides</strong>, it was concluded that in contrast to some reported cases [11–16], there is a very significant difference in binding when comparing the binding to selected protein segments or to the whole protein. Although <strong>studies</strong> using <strong>peptides</strong> are very relevant, clearly in the case <strong>of</strong> the interaction between the bafilomycin A 1 or SB 242784 <strong>with</strong> the c-subunit from V-ATPase, the whole protein architecture and the environment that it provides, are key factors for an efficient binding. Acknowledgments This work was supported by contract no. QLG-CT-2000- 01801 <strong>of</strong> the European Commission (MIVase consortium). M. H. and M. P. are members <strong>of</strong> the COST D22 Action <strong>of</strong> the European Union. F. F. acknowledges grant SFRH/BD/ 14282/2003 from FCT (Portugal). This work was partially funded by FCT (Portugal) under the program POCTI. References [1] M.E. Finbow, M.A. Harrison, The vacuolar H+-ATPase: a universal proton-pump <strong>of</strong> eukaryotes, Biochem. J. 324 (1997) 697–712. [2] T. Nishi, M. Forgac, The vacuolar (H+)-ATPases—Nature's most versatile proton pumps, Mol. Cell. Biol. 3 (2002) 94–103. [3] B. Powell, L.A. Graham, T.H. Stevens, Molecular characterization <strong>of</strong> the yeast vacuolar H+-ATPase proton pore, J. Biol. Chem. 275 (2000) 23654–23660. [4] M.A. Harrison, M.E. Finbow, J.B. Findlay, Postulate for the molecular mechanism <strong>of</strong> the vacuolar H(+)-ATPase (hypothesis), Mol. Membr. Biol. 14 (1997) 1–3. [5] B.J. Bowman, E.J. Bowman, Mutations in subunit c <strong>of</strong> the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site, J. Biol. Chem. 277 (2002) 3965–3972. [6] G. Werner, H. Hagenmaier, H. Drautz, A. Baumgartner, H. Zähner, Metabolic products <strong>of</strong> microorganisms. Bafilomycins, isolation, chemical structure and biological activity, J. Antibiot. 37 (1984) 110–117. [7] M. Harrison, B. Powell, M.E. Finbow, J.B.C. Findlay, Identification <strong>of</strong> lipid-accessible sites on the Nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H+-ATPase: site-directed labeling <strong>with</strong> n-(1-pyrenyl) cyclohexylcarbodiimide and fluorescence quenching analysis, Biochemistry 39 (2000) 7531–7537. [8] T. Páli, G. Whyteside, N. Dixon, T. Kee, S. Ball, M.A. Harrison, J.B.C. Findlay, M.E. Finbow, D. Marsh, <strong>Interaction</strong> <strong>of</strong> inhibitors <strong>of</strong> the vacuolar H+-ATPase <strong>with</strong> the trans<strong>membrane</strong> Vo-sector, Biochemistry 43 (2004) 12297–12305. [9] E.J. Bowman, L.A. Graham, T.H. Stevens, B.J. Bowman, The bafilomycin/concanamycin binding site in subunit c <strong>of</strong> the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae, J. Biol. Chem. 279 (2004) 33131–33138. [10] M. Harrison, J. Murray, B. Powell, Y. Kim, M.E. Finbow, J.B.C. Findlay, Helical interactions and <strong>membrane</strong> disposition <strong>of</strong> the 16-kDa proteolipid subunit <strong>of</strong> the vacuolar H1-ATPase analyzed by cysteine replacement mutagenesis, J. Biol. Chem. 274 (1999) 25461–25470. [11] T.L. Lentz, V. Chaturverdi, B.M. Conti-Fine, Amino acids <strong>with</strong>in residues 181–200 <strong>of</strong> the nicotinic acetylcholine receptor alpha 1 subunit in nicotine binding, Biochem. Pharmacol. 55 (1998) 341–347. [12] P.T. Wilson, T.L. Lentz, E. Hawrot, Determination <strong>of</strong> the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit <strong>of</strong> the acetylcholine receptor from Torpedo californica, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 8790–8794. [13] D. Neumann, D. Barchan, M. Fridkint, S. Fuchs, Analysis <strong>of</strong> ligand binding to the synthetic dodecapeptide <strong>of</strong> the acetylcholine receptor a subunit, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 9250–9253. [14] A.H. Taylor, G. Heavner, M. Nedelman, D. Sherris, E. Brunt, D. Knight, J. Ghrayeb, Lipopolysaccharide (LPS) neutralizing <strong>peptides</strong> reveal a lipid A binding site <strong>of</strong> LPS binding protein, J. Biol. Chem. 270 (1995) 17934–17938.
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UNIVERSIDADE TÉCNICA DE LISBOA INS
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Fernandes, F., Loura, L. M. S., Fed
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Ao Pedro e Hugo, amigos de longa da
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2.2. - Peptides as models 2.3. - Am
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ABBREVIATIONS AND SYMBOL LIST ABBRE
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RESUMO RESUMO As biomembranas são
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SINOPSE SINOPSE Nas últimas duas d
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SINOPSE podem fornecer informação
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SINOPSE aceitantes. Dadores mais pr
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OUTLINE OUTLINE The last two decade
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OUTLINE membranes with a distributi
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OUTLINE BAR domains (tubulation of
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ion diffusion, as the energy requir
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acid. If no more groups are linked
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sphingomyelin, the most abundant sp
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signal for neighbouring cells to ph
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functional role in process such as
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in the L α phase, while lateral di
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1.7. Lateral heterogeneity in lipid
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the vesicle through bilayer deforma
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Figure I.9 - Depiction of the sever
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Figure I.11 - Experimentally obtain
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drying into a film and ressuspensio
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deactivating agents. Zwitterionic d
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amphipatic helices (see Section 2.3
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size corresponding to the hydrophob
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changes abruptly in the interfacial
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thickness of the bilayer (Section 1
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some α-helical membrane proteins a
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The formation of a lipid population
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homogeneous distribution of lipids
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Figure I.20 - Relative binding cons
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2.9. Lipid phase preferential parti
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In Figure I.21, theoretical simulat
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PROTEIN-PROTEIN AND PROTEIN-LIPID I
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PROTEIN-PROTEIN AND PROTEIN-LIPID I
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PROTEIN-PROTEIN AND PROTEIN-LIPID I
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19. Fernandes, F., L. M. S. Loura,
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Figure Legends Fig.1: N-terminal am
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FIG. 1A FIG.1B 17
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FIG. 3A FIG. 3B 19
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FIG.5A 21
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FIG 6. 23
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FIG. 8 A B 25
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The signalling functions of PI(4,5)
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174
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zoxadiazol (NBD)-PC were obtained f
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Fig. 3. Fluorescence decays of diph
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CONCLUSIONS VII CONCLUSIONS Chapter
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CONCLUSIONS constants recovered by
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CONCLUSIONS Chapter IV ‐ Binding
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CONCLUSIONS Chapter VI - Clustering
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FINAL CONSIDERATIONS AND PROSPECTS
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BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
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BIBLIOGRAPHY Phosphatidylinositol 4
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BIBLIOGRAPHY Glaubitz, C., Grobner,
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BIBLIOGRAPHY Koehorst, R. B. M., Sp
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BIBLIOGRAPHY Mishra, V. K., Palguna
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BIBLIOGRAPHY Pluschke, G., Hirota,
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BIBLIOGRAPHY Sperotto, M. M., and M
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BIBLIOGRAPHY Williamson, I. M., Alv