10 A niversary of IIMCB

• Jozwiak K, Krzysko KA, Bojarski L, Gacia M, Filipek S.
Molecular models of the interface between anterior
pharynx-defective protein 1 (APH-1) and presenilin
involving GxxxG motifs. ChemMedChem, 2008; 3:627-634
• Majewski T, Lee S, Jeong J, Yoon DS, Kram A, Kim MS,
Tuziak T, Bondaruk J, Lee S, Park WS, Tang KS, Chung W,
Shen L, Ahmed SS, Johnston DA, Grossman HB, Dinney
CP, Zhou JH, Harris RA, Snyder C, Filipek S, Narod SA,
Watson P, Lynch HT, Gazdar A, Bar-Eli M, Wu XF, McConkey
DJ, Baggerly K, Issa JP, Benedict WF, Scherer SE, Czerniak
B. Understanding the development of human bladder
cancer by using a whole-organ genomic mapping
strategy. Lab Invest, 2008; 88:694-721
Current Research
1. Studies of activation switches in opioid receptors
G protein coupled receptors (GPCRs) interact with very
diverse sets of ligands which bind to the transmembrane
(TM) segments and sometimes also to the receptor
extracellular domains. Each receptor subfamily undergoes
a series of conformational rearrangements leading to
the binding of a G protein during the activation process.
All GPCRs preserved the 7-TM scaffold during evolution
but adapted it to different sets of ligands by structure
customization. Binding of structurally different agonists
requires the disruption of different intramolecular
interactions, leading to different receptor conformations and
differential effects on downstream signaling proteins. The
dynamic character of GPCRs is likely to be essential for their
physiological functions, and a better understanding of this
molecular plasticity could be important for drug discovery.
Experiments suggest that agonist binding and receptor
activation occur through a series of conformational
intermediates. Transition between these intermediate states
involves the disruption of intramolecular interactions that
stabilize the basal state of a receptor. Such profound changes
are evoked by the action of molecular switches. The switches
proposed so far for different GPCRs include the “rotamer
toggle switch” involving the CWxPxF sequence on helix TM6,
the switch based on the NPxxY(x)(5,6)F sequence linking
helices TM7 and H8, the “3-7 lock” interaction connecting
TM3 and TM7 (involving the Schiff base-counterion
interaction in rhodopsin), and the “ion lock” linking
transmembrane helices TM3 and TM6 and employing the
E/DRY motif on TM3. Although, in the rhodopsin structure,
all these switches are closed (inactive state), in recent crystal
structures of b 1 - and b 2 -adrenergic receptor complexes with
antagonists and inverse agonists the “ion lock” is open while
the “rotamer toggle switch” remains closed.
Opioid receptors belong to the family of GPCRs. They
are located in the membranes of neurons of the central
nervous system and of some types of smooth muscle cells.
Due to the important role they play in the human body in
controlling pain and stress, modulating immune responses
and developing addiction, opioid receptors have been the
subject of numerous investigations. There are four types of
opioid receptors: µOR, δOR, κOR and the nociceptin/opioid
receptor-like 1. There are also additional, pharmacologically
classified, subtypes of opioid receptors, though it is
believed that they may, at least partly, originate from
homodimerization of the four main opioid receptor types
and their heterodimerization with other GPCRs. Knowledge
of the structural details of receptor activation is absolutely
necessary in order to design new drugs with precise action
and negligible side effects.
Opioid receptors, like other GPCRs, undergo specific
structural rearrangements upon activation by agonists.
Such processes proceed via several steps ruled by different
molecular switches. The first event in opioid receptor
activation includes sensing of agonists and antagonists.
Agonist binding is the first step in ligand-induced receptor
activation. To investigate the relationship between the
final movements of a ligand in a receptor binding site and
the first steps of the activation process in opioid receptors,
we chose a set of rigid ligands with the structural motif
of tyramine (p-hydroxyphenethylamine) so that the two
parts - the “message” (tyramine) and the “address” - are
well distinguished. We used antagonists - naltrexone and
b-FNA, and two closely related agonists - morphine and
N-methyl-morphine.
Using homology modeling, simulated annealing and
molecular dynamics of the µ opioid receptor complexes,
we proposed distinct binding modes of opioids carrying
the same structural motif – tyramine. Although they
bind to the same binding pocket and the protonated
amine interacts with D3.32, the antagonist’s phenolic OH
group tends to bind Y3.33 whereas the agonist’s phenolic
OH group tends to bind H6.52 (numbers according to
the Ballesteros-Weinstein numbering scheme). All the
studied agonists broke the “3-7 lock” (the hydrogen
bond D3.32-Y7.43 between TM3 and TM7). Moreover, the
antagonist naltrexone, when restrained to bind H6.52,
was also able to break this connection (Fig. 1a,b) and,
Fig. 1. The structure of
naltrexone-µOR complex
from unrestrained (ligand
in green) and restrained
(ligand in orange) MD
simulations. During the
restrained simulation
(a bond between the
phenolic OH group of the
ligand and H6.52 was fixed) the “3-7 lock” (a hydrogen bond D3.32-Y7.43
between TM3 and TM7) is breaking. (a) view from the extracellular side.
(b) a side view of the same structure (author: Sławomir Filipek).
Laboratory of Biomodelling 49