1 Department of Neurochemistry, Chemical Institute, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri út 5967, H-1025 Budapest, Hungary
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Abstract |
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Keywords: 3D pharmacophore/lysine switch/rational drug design/stabilization centres
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Introduction |
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If the transmembrane channel of the native receptor is gated by the agonist-induced domain closure, and if structural changes proceed from local ligand binding to global domain closure, a local indicator might be used to predict the ligand function. We think the Lys-switch is such an indicator, and therefore its position might predict the agonist or the antagonist character of native AMPA receptor ligands in general.
The aim of this note is to offer a simple in silico method to determine the agonist or antagonist character of new ligands for this receptor. The in silico approach adds to the drug discovery toolbox the value of fast and significant reduction in the number of new molecules to synthesize and/or to test.
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Materials and methods |
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To investigate the Lys730-switch, the following procedure was followed. In the apo, DNQX-in-DNQX, AMPA-in-AMPA, Glu-in-Glu, and kainate-in-kainate structures, the NH3+ group of Lys730 was rotated around its last CC bond in increments of 10°, and the total energy of the system was calculated at each torsion angle. Several minima were found this way, in each of which an additional energy minimization was done with unconstrained protein and ligand geometry. From the resulting configurations, the minimum with the lowest energy was taken as the most probable conformation, and the position of the Lys730-switch was determined (i.e. whether Lys730 was hydrogen-bonded to Asp728 or to Glu705). The energies of the Lys730Asp728 and the Lys730Glu705 configurations differed by approximately 620 kcal/mol.
Since ligands react with unoccupied binding sites, similar calculations were done by placing various ligands in the apo iGluR2 S1S2 structure. Ligands were initially positioned by structure fitting. Taking the Glu-in-apo structure as an example, respective atoms of the seven residues forming the binding cleft (Tyr450, Pro478, Thr480, Arg485, Ser654, Thr655 and Glu705) were fitted in a RMS sense, that is the relaxed Glu-in-Glu and apo structures were approximately superimposed and the ligand at the appropriate position was transferred to the apo structure. The ligands for which no experimental X-ray complex structure is available were placed by using isosteric analogies: the zwitterionic part of (S)F-willardiine was aligned with that of (S) AMPA and the condensed rings of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) with those of DNQX (see Figure 1). In the unconstrained minimization procedure (neither the protein nor the ligand geometry was fixed) the global protein backbone structure remained essentially identical to that of the apo structure even with agonists, i.e. MM calculations were found to be unable to reproduce domain closure. This is to be expected since MM energy minimization searches for a local minimum and the large scale backbone rearrangement probably involves initial and final states separated by quite a few of the local energy maxima which are impossible to pass for the energy minimization algorithms.
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SC residues are defined in the following way (see Figure 2): those residues, which are at least 10 residues away in the amino acid sequence, but contact each other are selected. If it is possible to select one residue from both flanking tetrapeptides of both residues so that at least seven contacts are made between the formed two triplets out of the theoretically possible nine, then the central residues are defined as SCs (Dosztányi et al., 1997
). (Two residues are considered to contact each other if they have atoms in a distance less than the sum of their van der Waals radii plus 1 Å.)
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Results and discussion |
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Furthermore, the Lys730-switch position found with () [S]-1-(2-amino-2-carboxyethyl)-5-fluoropyrimidine-2,4-dione [(S)F-willardiine], for which no experimental crystal structure is available, indicates that (S)F-willardiine behaves as an agonist. Indeed, this molecule is known to be (Stensbøl et al., 1999) a full agonist at the AMPA receptor. This shows that prediction of the agonist character of a new ligand might be possible by determining the Lys730-switch position in the apo structure.
Q5 was recently shown (Szárics et al., 2001) to be a novel AMPA-receptor subtype-specific antagonist. The Lys730-switch position (Lys730 to Glu705) is in accordance with the character found experimentally (Szárics et al., 2001
).
The proposed simple method relies on two key assumptions. First, we assume that the open conformation of the LBC (observed in the apo and antagonist-bound crystalline states) corresponds to the resting state (closed channel), and ligand binding takes place in the apo structure. Secondly, we assume that (i) the conformational changes leading to channel opening are agonist-induced and (ii) local conformational changes initiated by agonist binding in the apo structure trigger a sequence of events leading to global changes (LBC closure) which, in turn, results in the active state of the native, wild-type receptor. Both assumptions are far from trivial, and will be discussed below.
X-ray diffraction studies on the 3D structure of the metabotropic glutamate receptor mGluR1 indicated that the smallest functional unit is a dimer (Kunishima et al., 2000; Tsuchiya et al., 2002
). These authors identified two, substantially different ligand-free conformations, one of which being similar to the Glu-liganded complex. The active or resting state of the receptor appears to be determined by the structure of the dimer interface, not by the closed or open state of a single subunit, which changes its conformation irrespective of the presence of the agonist. Thus, domain closure appears to be a necessary but not a sufficient condition for the receptor to turn active. While the openopen dimer (formed spontaneously or wedged by the antagonist) is probably always in the resting state, the openclosed and closedclosed dimers can either be in the resting or in the active state depending on the status of the dimer interface. Nevertheless, the authors leave open the question of the receptor activation mechanism, i.e. whether agonist binding stabilizes the spontaneously active forms out of the multiple conformations in equilibrium or induces the resting-to-active transition. However, since agonist binding was observed in both the open and closed LBC, we think that our assumptions cannot be justified, and the proposed method cannot be used for in silico prediction of the mGluR1 activation.
For the prokaryotic iGluR0 receptor it was proposed that agonist-induced conformational changes cause the ion channel to open, leaving open the question whether this holds for iGluR2 since prokaryotic and eukaryotic iGluRs bind ligands via different mechanisms (Mayer et al., 2001). In a previous binding and electrophysiological study on iGluR0 (Chen et al., 1999
), however, conservation of the binding core residues was considered to indicate that the chemical nature of the gate and the mechanism by which it is opened and closed may be similar in all iGluR channels. Until further, more specific details emerge, we cannot decide whether our assumptions are valid or not for the prokaryotic iGluR0 gating mechanism.
For the eukaryotic iGluRs containing iGluR2 subunits, however, the assumptions appear to be valid. In contrast to the mGluR1 case, structural data (Armstrong and Gouaux, 2000) show the presence of a single apo structure and there is no indication of spontaneous LBC closure. Consequently, ligands bind initially in the apo state. Antagonists wedge the LBC preventing further changes, while the emerging ligandprotein interactions on agonist binding trigger a series of conformational changes leading to LBC closure and receptor activation.
Recent work on iGluR2 mutants (Sun et al., 2002) further supports the agonist-induced LBC closure and receptor activation mechanism, although the smallest functional unit being the dimer, relaxation of the dimer interface may lead to an inactive (desensitized) state with a closed LBC. We note that the proposed desensitization scheme is not entirely consistent with the bulk of experimental data analysed by Kardos and Nyikos (Kardos and Nyikos, 2001
). Nevertheless, since desensitization, by whatever mechanism, is a phenomenon observed with agonists only, the predictability of agonist function remains possible.
Based on the above discussion, we might safely assume that the initial steps of ligand binding take place in the apo structure. Since Glu705 is proximal to the binding pocket, and all agonists studied enter into direct interaction with this residue (see Table I), we propose that agonist binding does activate the Lys-switch by forcing Lys730 to Asp728. Since the calculations indicate that the switch assumes its final position even when global closure is absent, we might conjecture that it is an early step in the chain of conformational changes leading finally to channel opening. We note, nevertheless, that if the Lys730-switch is used as a diagnostic tool only, it is irrelevant whether switch operation precedes or follows other conformational changes.
Based on SC analysis, we offer additional evidence on the strategic location of the Lys-switch. In general, the iGluR2 structures show striking SC uniformity although the global 3D structures are quite different for the resting and complexed states. The residue pairs forming SCs in the case of the 1FTJ structure are shown in Figure 3. A closer look at the SCs identified in iGluR2 S1S2 reveals that two key residues close to the binding cleft, Thr480 and Tyr732, form SCs in all structures whereas Pro478, Arg485 and Ser654 do not. The residue Leu650 forms a SC with Phe682 in the experimental apo, DNQX and kainate, but not in the full agonist structures. The finding is remarkable, because Armstrong and Gouaux (Armstrong and Gouaux, 2000
) quote the position of Leu650 as the major difference in Glu and Kai (kainate), binding discriminating full versus partial agonist behaviour. In addition, the SC involving Ile500 and Leu727 in the experimental iGluR2 S1S2 complexes with agonists (Glu, AMPA and Kai) is absent in the experimental iGluR2 S1S2 complexed with the antagonist (DNQX). These amino acid residues participate in the backbone torsion leading to the shell closure (Armstrong and Gouaux, 2000
). The residue Ile500, in turn, forms SCs with Leu704, Glu705 and Asp728: two of them being members of the Lys-switch. The Lys-switch and its neighbourhood (Lys730 forms SCs with Leu498 and Gly499) sit in a stable island (indicated by an arrow in Figure 3
) and, also, they are close to the binding cleft, the hinge region, and to the point where backbone torsion startsthis might explain why the Lys730-switch plays the key role. The uniformity of the SC maps suggests that a simple activation mechanism is operating and the close proximity of the hinge region and the ligand binding site allows one to conjecture that the sequence of conformational changes is indeed ligand-induced, and the Lys-switch operation is indeed an early step in the sequence of events.
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The present application of SC analysis for a receptor ion channel suggests that it is a powerful tool of identifying the residues important in ligand binding and subsequent structural transitions.
One may ask whether these results are specific to the iGluR2 S1S2 LBC or may be more generally valid. Since the experimental 3D structures of other iGluRs are unknown, we cannot tell as yet. However, it should be noted that in the Ligand Gated Ion Channel Database (Le Novère and Changeux, 2001), out of the 52 sequence-aligned homologous iGluRs the residues Glu705, Asp728 and Lys730 (denoted there as E1038, D1064 and K1066, respectively) are conserved in 28 cases (54%). This finding might perhaps substantiate a Lys-switch-like mechanism at work. In 12 NMDA2 receptors (23%), these residues are markedly different, meaning that either ligand-gating occurs by a different mechanism or local structural changes near to the binding cleft other than the Lys-switch might operate here.
As located in a strategic place of the iGluR2 S1S2, the change in the position of the Lys730-switch is proposed to act as a sign to show the way to either the resting or to the complexed (active and/or desensitized) ligand-specific conformations. In silico prediction of the agonist or antagonist character of a new AMPA receptor ligand might therefore be possible in combination with in vitro binding studies. The diagnostic potential of the Lys730-switch substantiates that agonist binding in iGluR2 does prime sequential changes into active receptor conformations that were not present a priori.
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Notes |
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Acknowledgments |
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References |
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Received December 27, 2001; revised May 24, 2002; accepted May 25, 2002.