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Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core

2003; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês

10.1093/emboj/cdg303

1460-2075

Hiroyasu Furukawa, Eric Gouaux,

Molecular Sensors and Ion Detection

Article16 June 2003free access Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core Hiroyasu Furukawa Hiroyasu Furukawa Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, NY, 10032 USA Search for more papers by this author Eric Gouaux Corresponding Author Eric Gouaux Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, NY, 10032 USA Search for more papers by this author Hiroyasu Furukawa Hiroyasu Furukawa Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, NY, 10032 USA Search for more papers by this author Eric Gouaux Corresponding Author Eric Gouaux Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, NY, 10032 USA Search for more papers by this author Author Information Hiroyasu Furukawa1 and Eric Gouaux 1 1Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, 650 West 168th Street, New York, NY, 10032 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2873-2885https://doi.org/10.1093/emboj/cdg303 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Excitatory neurotransmission mediated by the N-methyl-D-aspartate subtype of ionotropic glutamate receptors is fundamental to the development and function of the mammalian central nervous system. NMDA receptors require both glycine and glutamate for activation with NR1 and NR2 forming glycine and glutamate sites, respectively. Mechanisms to describe agonist and antagonist binding, and activation and desensitization of NMDA receptors have been hampered by the lack of high-resolution structures. Here, we describe the cocrystal structures of the NR1 S1S2 ligand-binding core with the agonists glycine and D-serine (DS), the partial agonist D-cycloserine (DCS) and the antagonist 5,7-dichlorokynurenic acid (DCKA). The cleft of the S1S2 ‘clamshell’ is open in the presence of the antagonist DCKA and closed in the glycine, DS and DCS complexes. In addition, the NR1 S1S2 structure reveals the fold and interactions of loop 1, a cysteine-rich region implicated in intersubunit allostery. Introduction N-methyl-D-aspartate (NMDA) receptors occupy a unique position amongst ligand-gated ion channels because they require both glycine and glutamate for activation, and membrane depolarization to relieve block by magnesium (Cull-Candy et al., 2001). The prerequisite for simultaneous chemical and electrical stimuli, and the subsequent influx of calcium through the ion channel, distinguish NMDA receptors from (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) and kainate ionotropic glutamate receptors (iGluRs) (Dingledine et al., 1999). Under normal circumstances, NMDA receptors are involved in activity-dependent synaptic plasticity (Lisman and McIntyre, 2001) and in learning and memory (Nakazawa et al., 2002). NMDA receptors are also implicated in a number of disease and injury states, including schizophrenia and excitotoxicity (Mohn et al., 1999; Tsai and Coyle, 2002). In fact, NMDA receptor agonists such as D-cycloserine (DCS) show promise in the treatment of individuals with schizophrenia and Alzheimer's disease (Kemp and McKernan, 2002). Consonant with the requirement of glycine and glutamate for activation of NMDA receptors, the intact receptor is a complex of four or five subunits that typically include the NR1 subunit together with the NR2A-D or NR3A-B subunits (Cull-Candy et al., 2001). NR1 comprises the glycine-binding subunit and the NR2A-D subunits possess the glutamate-binding site (Hollmann, 1999). Interestingly, NR3A-B subunits can combine with NR1 upon expression in Xenopus oocytes to form receptors that are solely activated by glycine (Chatterton et al., 2002). The architecture of NMDA receptors is similar to non-NMDA receptors: each NMDA receptor subunit has three transmembrane segments (M1, M2 and M3), a re-entrant membrane loop (P loop), an extracellular N-terminus and an intracellular C-terminus (Figure 1B). Like the other iGluR subtypes, the agonist-recognition region of an NMDA receptor subunit is defined by polypeptide segments S1 and S2 (Stern-Bach et al., 1994). Inspection of an alignment of the S1 and S2 regions of iGluRs, however, demonstrates that loop 1 of NMDA receptors is cysteine rich and ∼30 residues longer than the corresponding regions of AMPA and kainate receptors. The N-terminal domain (ATD) is defined by the first ∼400 amino acid residues and, while it is not directly involved in agonist binding, it is implicated in subunit assembly (Perez-Otano et al., 2001) and in receptor modulation by protons, polyamines, Zn2+ and ifenprodil (Zheng et al., 2001). The intracellular C-terminal domain (CTD) is a localization and regulatory module, and it interacts with numerous post-synaptic molecules. Figure 1.(A) Chemical structures of NMDA NR1 ligands. (B) Domain organization of a NR1 subunit showing the S1 and S2 segments in light blue and pink, respectively. The N-terminal domain (ATD), transmembrane segments and C-terminal domain (CTD) are not included within the S1S2 construct. (C) Multiple sequence alignment of S1 and S2 segments from rat NMDA, AMPA and kainate receptors. DDBJ/EMBL/GenBank accession Nos: X63255 (NR1), M91561 (NR2A), M91562 (NR2B), M91563 (NR2C), L31611 (NR2D), AF073379 (NR3A), AF440691 (NR3B), M85035 (GluR2), M85037 (GluR4) and Z11548 (GluR6). Drawn above the aligned sequences is the secondary structure determined from the NR1 S1S2 glycine structure where α-helices and β-strands are represented as rectangles and arrows, respectively. The color of the S1 and S2 segments is the same as that used in (B). Dots indicate the region where no electron density for the main chain is available. Cysteine residues participating in disulfide bond formation (green circles) are connected to their partners by green lines. Residues directly involved in agonist binding are marked with orange stars, whereas those specifically involved in antagonist binding are marked with blue + symbols. The sequences are numbered according to the predicted mature and immature polypeptides for non-NMDA and NMDA receptors, respectively. Download figure Download PowerPoint Structural and functional studies of GluR2 S1S2 have yielded insights into the pharmacology and mechanism of desensitization (Armstrong and Gouaux, 2000; Jin et al., 2002; Sun et al., 2002). However, structural studies on NMDA receptors have lagged behind, to some extent, because of the difficulty in obtaining milligram quantities of pure functional protein. Nevertheless, several groups have performed molecular modeling studies of the NMDA receptor S1S2 (Laube et al., 1997; Tikhonova et al., 2002). While modeling studies can provide important insights, they are limited when conformational changes accompany ligand binding and when solvent molecules mediate key interactions. Moreover, the structure of loop 1, as well as subunit–subunit interaction surfaces and modes of association, are currently without precedent. Therefore, the structure of the NMDA receptor ligand-binding core, its modes of antagonist, partial agonist and full agonist binding, and its conformational states are unknown. Here we report the structures of the rat NR1 S1S2 ligand-binding core in complexes with two physiological full agonists, glycine and D-serine (DS), the partial agonist DCS and an antagonist, 5,7-dichlorokynurenic acid (DCKA). Results Preparation and ligand-binding activity of NR1 S1S2 The boundaries of the NR1 S1S2 construct were based on GluR2 S1S2 (Figure 1). After removal of the His tag, the amino acid sequence begins with GM394S395T396..., where the native NR1 sequence starts at Met394 and ends with Ser800; a GT linker connects the S1 and S2 segments. In contrast to previous studies of NMDA receptor S1S2 constructs expressed in insect cells (Ivanovic et al., 1998; Miyazaki et al., 1999), the construct reported here is monomeric, as judged by size exclusion chromatography (SEC) and sedimentation equilibrium experiments at concentrations up to 1 mg/ml (data not shown). The recombinant NR1 S1S2 showed specific and saturable binding of the glycine-site antagonist [3H]MDL105,519 (Figure 2). The [3H]MDL105,519 binding was displaced by glycine, DS, DCS and DCKA (Figure 2B), and the measured Kd and Ki values were similar to those reported for an S1S2 construct or a full-length receptor expressed in insect cells (Ivanovic et al., 1998; Miyazaki et al., 1999). Figure 2.Ligand-binding properties of NR1 S1S2 as assessed by (A) saturation and (B) displacement experiments using the competitive antagonist of [3H]MDL105,519. The measured Kd value for [3H]MDL105,519 is 5.86 nM and the Ki values are 26.4 μM (glycine), 7.02 μM (DS), 241 μM (DCS), 0.54 μM (DCKA) and 2.30 mM (L-serine). Download figure Download PowerPoint NR1 ligand-binding core structure and glycine-binding site The glycine-bound NR1 S1S2 structure unambiguously reveals a bilobed or ‘clamshell’ structure consisting of domains 1 and 2 (Figure 3). Following model building and refinement, there was clear density for 281 out of 292 residues in the S1S2 construct. Three residues at the N-terminus of S1 and eight residues in loop 1 (Asp441–Arg448) were disordered. The fold of the NR1 S1S2 is similar to that of GluR2 S1S2 (Armstrong et al., 1998) even though the level of amino acid sequence identity is low (27%). Following superposition, the root-mean-square (r.m.s.) deviation on Cα positions between GluR2 S1S2 and NR1 S1S2 is 1.02 Å, where loops 1 and 2 and helix G of NR1 S1S2 and the corresponding regions of GluR2 S1S2 were excluded from the calculation. Figure 3.Structure of glycine-bound NR1 S1S2. (A) Ribbon representation of the glycine-bound state with S1 and S2 colored as in Figure 1B and viewed from the side. Glycine binds in the crevice between domains 1 and 2, and is surrounded by Pro516, Thr518, the N-terminal regions of helices D, F and H, and β-strand 14; residues from both domain 1 and domain 2 make contacts with the α substituents of glycine. The three disulfide bonds in NR1 S1S2 (Cys420–Cys454, Cys436–Cys455 and Cys744–Cys798) are drawn as green lines. The first two are in loop 1 and the last one is near the C-terminus (CT). (B) Ribbon representation of domain 1 viewed from the top of the N-terminus (NT). Protruding as far as 15 Å from domain 1 are loops 1 and 2. The disulfide bonds (Cys420–Cys454 and Cys436–Cys455) drawn as green lines are helping to knit together the β-strands and loop regions of loop 1. (C) Schematic representation of the loop 1 region. The dashed line indicates the region (Pro441–Arg448) where no electron density for the main chain is available. Download figure Download PowerPoint The greatest structural difference between NR1 and GluR2 S1S2 is in loop 1, which contains a pair of antiparallel β-strands and intervening loops that are bound by two disulfide bridges. Interestingly, there is clear electron density for an alternate conformation for the side chain of Cys454 which would preclude formation of a disulfide bridge with Cys420. The length of loop 1, in conjunction with an extended loop 2, make domain 1 of NR1 ∼15 Å wider than GluR2 and GluR0 (Mayer et al., 2001). More specifically, strands 1–5 form a protein ‘wall’ projecting from domain 1. Loop 2 forms a pair of antiparallel β-strands projecting from domain 1, as in GluR2 S1S2. As with all eukaryotic iGluRs, there is a disulfide bond in NR1 located between Cys744 on helix I and Cys798 at the C-terminus of S2, linking the end of helix K on domain 1 with domain 2. Glycine binds in the domain 1–domain 2 crevice and is surrounded by the N-terminus of helix D, helix F, helix H and β-strand 14. Residues from domains 1 and 2 make contacts with the α substituents of glycine through eight direct hydrogen bonds and electrostatic interactions (Figure 4B). In addition, water molecules W1, W2, W4 and W5 form interactions between glycine and NR1 S1S2. The α-carboxy group of glycine makes an essential interaction with the guanidinium group of Arg523, a residue conserved among all iGluRs. In GluR2 S1S2, the corresponding arginine residue interacts with the α-carboxy group of glutamate (Armstrong and Gouaux, 2000). The α-carboxy group of glycine also hydrogen bonds to the backbone amide groups of Thr518 and Ser688, and to the hydroxyl group of Ser688. The positively charged amino group of glycine interacts with the carbonyl oxygen of Pro516, the hydroxyl group of Thr518 and the carboxylate oxygen of Asp732, which is either an aspartate or a glutamate residue in iGluRs. Gln405, which is near the bound glycine, interacts with two residues in domain 2: a direct hydrogen bond to the indole nitrogen of Trp731 and, via W3, a water-mediated hydrogen bond to Asp732. Figure 4.The mechanism of glycine binding. (A) An Fo − Fc ‘omit’ electron density map using data to 1.35 Å resolution where the atoms corresponding to glycine, selected ligand-binding residues and waters W1, W2 and W3 were omitted from the Fc calculation. The contour level is 4.2 σ. (B) Stereo view of glycine (black bonds) and the interacting residues (yellow bonds). Dashed lines indicate hydrogen bonds and ionic interactions (interatom distance <3.2 Å). Water molecules (cyan) make important contributions to the hydrogen bond network and stabilize the binding of glycine. Download figure Download PowerPoint Structural distinction between NR1 S1S2 and GluR2 S1S2 There are significant differences between the NR1 S1S2 and GluR2 S1S2 structures, even though their overall folds are similar (Figure 5). First, when comparing NR1 S1S2 and GluR2 S1S2 in their respective complexes with full agonists, NR1 S1S2 adopts a more closed conformation. Secondly, loop 1 in NR1 has a more substantial and complex structure in comparison with loop 1 in GluR2. Thirdly, loop 2 in NR1 S1S2 is longer and protrudes farther from domain 1 in comparison with loop 2 of GluR2. Lastly, the orientation of helix G, relative to the remainder of domain 2, is different in NR1 S1S2 (Figure 5). Figure 5.Superposition of the glycine-bound NR1 S1S2 (light cyan) and the L-glutamate-bound GluR2 S1S2 (light coral) structures using only Cα atoms viewed from (A) the side and (B) the top of the N-terminus. (C) Stereo view of the same superposition structures at the ligand-binding pocket with glycine (black) and L-glutamate (gray) interacting with the residues from NR1 S1S2 (cyan) and GluR2 S1S2 (crimson), respectively. The specificity of NR1 for glycine can be explained by (i) the hydrophobic environment created by Val689 and Trp731 and (ii) a steric constraint caused by the positioning of Trp731, which in GluR2 is Leu704 pointing away from the binding pocket, which disallow the γ-carboxyl group of L-glutamate to reside. Download figure Download PowerPoint The essential functional difference between NR1 and other iGluR subtypes is that NR1 has a high affinity for glycine and an unmeasurably low affinity for L-glutamate (Miyazaki et al., 1999). To understand the agonist selectivity of NR1, we have superimposed NR1 S1S2 and GluR2 S1S2 (Figure 5C). Strikingly, the binding site residues of NR1 and GluR2 S1S2 superpose well and most of the agonist-contacting residues are identical or are conservative substitutions (NR1/GluR2 = Pro516/478, Thr518/480, Arg523/485, Ser688/654 and Asp732/Glu705) and share similar orientations. However, there are two critical differences between the NR1- and GluR2-binding sites. Residue 655 in GluR2 is a threonine and the hydroxyl group makes a hydrogen bond to a γ-carboxylate oxygen of glutamate; in NR1, the equivalent residue is Val689, which cannot form a similar interaction. The second key difference is residue Trp731 in NR1, which in GluR2 is Leu704. The indole ring in NR1 faces the agonist-binding pocket and, in the context of GluR2, would clash with the γ-carboxylate of glutamate. In GluR2, the leucine not only takes up less volume, but it is also oriented differently, thus allowing for the binding of the γ-carboxylate of glutamate. In NR2 subunits, the equivalent residue is a tyrosine. We suggest that the local hydrophobic environment created by Val689 and Trp731, as well as the steric constraint caused by the orientation of Trp731, prevents the binding of the γ-carboxyl group of L-glutamate. D-serine and D-cycloserine complexes The conformation of NR1 S1S2 in the DS and DCS complexes is similar to the S1S2 conformation in the glycine-bound form and the three complexes have essentially the same degree of domain closure. Indeed, the glycine, DS and DCS cocrystals are all isomorphous. Superpositions of α–carbon atoms of the DS and DCS S1S2 structures with the glycine structure yield r.m.s. deviations of 0.10 and 0.20 Å, respectively. At the agonist-binding pocket, however, there are similarities and differences between the three agonists that are clearly defined by their respective electron densities. In the case of DS, the important differences are localized to the hydroxyl group as the α substituents are bound similarly to the α groups of glycine, with only one exception (Figure 6A). There are 10 salt-link and hydrogen-bonding interactions between DS and NR1 S1S2, along with interactions with two water molecules that form a hydrogen-bond network with residues Thr518, Ser688 and Asp732. At the α-carboxylate of DS, the hydroxyl of Ser688 is pointed away from the binding pocket and cannot make a hydrogen bond to an α-carboxylate oxygen, as it does in the glycine complex. Nevertheless, the DS hydroxyl group forms hydrogen bonds with the hydroxyl groups of Thr518 and Ser688 and the carboxyl group of Asp732. Figure 6.The binding mechanisms of DS and DCS. Stereo view of (A) DS and (B) DCS and interacting residues. In both cases, dashed lines indicate the potential hydrogen bonds and ionic interactions (interatom distance <3.2 Å). Water molecules (cyan), located in the binding pocket, are also forming a critical hydrogen-bond network to stabilize the binding of both DS and DCS. Download figure Download PowerPoint DCS binds similarly to DS, even though the cyclic agonist contains a unique functional group. In the DCS complex, the exocyclic oxygen and the nitrogen mimic the α-carboxylates of glycine and DS, interacting with the guanidinium group of Arg523 (Figure 6B). The isoxazolidinone ring oxygen, instead of an α-carboxylate oxygen in glycine, hydrogen bonds with Ser688. The α-amino group of DCS follows the same pattern of interactions as the α-amino groups of glycine and DS. An open-cleft conformation is stabilized by antagonist The NR1–DCKA cocrystals contain two molecules in each asymmetric unit with an expanded cleft between domain 1 and domain 2, relative to the complexes with full and partial agonists (Figure 7). The two molecules in the asymmetric unit are not identical in terms of domain closure, however, and they differ by ∼6° with molecule A being the most open. Besides the degree of domain separation, there are no large differences between the structures of molecules A and B including the orientation of the residues and water molecules surrounding the DCKA-binding pocket. The mechanism of DCKA binding is the same for both molecules. Relative to the NR1 S1S2 glycine structure, molecule A of the DCKA complex is 24° more open, i.e. there is 24° of domain closure in going from molecule A of the DCKA structure to the glycine complex. Figure 7.The binding mechanisms of DCKA. (A) Ribbon representation of the DKCA-bound NR1 S1S2 structure (DCKA molecule A) with S1 and S2 colored as in Figure 1B. (B) Superposition of the two DCKA-bound NR1 S1S2 molecules in an asymmetric unit (DCKA molecules A and B in blue and light blue, respectively) and the glycine-bound molecule (red). The superposition was calculated using Cα atoms. The r.m.s. deviation for DCKA molecules A and B is 0.74 Å. The glycine-bound form has a bilobed structure closed by 23.8° and 18.2° compared with the DCKA molecules A and B, respectively. (C) Stereo view of DCKA and interacting residues. Download figure Download PowerPoint DCKA binds primarily to the ‘upper’ side of the binding pocket and makes the largest number of direct interactions with residues from domain 1. Like agonists, the carb oxylate of DCKA forms a salt link with Arg523 and a hydrogen bond with the amino group of Thr518, while the amino group of DCKA forms a hydrogen bond with the main-chain carbonyl oxygen of Pro516. The quinoline ring of DCKA is ∼3.5 Å below the aromatic ring of Phe484, participating in a π-stacking interaction (Figure 7C) as predicted by modeling studies (Tikhonova et al., 2002). The chlorine atoms at the 5 and 7 position of DCKA are in van der Waals contact with the aromatic rings of Phe408 and Trp731, respectively. The carbonyl oxygen of DCKA, while not directly interacting with the protein, does form a hydrogen bond with a water molecule at the base of helix F. DCKA acts like a wedge between Gln405 and Trp731/Asp732 by occupying the space between the residues. Hydrogen bonds between the amide oxygen of Gln405 and the indole nitrogen of Trp731, together with a water-mediated (W3) contact between Gln405 and Asp732, are maintained in the full and partial agonist-bound states. However, these interdomain contacts are disrupted by DCKA. Discussion Native NMDA receptors are composed of subunits with distinct ligand-recognition properties and are modulated by a large number of effector molecules and various pre- and post-translational modifications. Thus, elucidating mechanisms of NMDA receptor function based on high-resolution structural information is a particularly important and as yet unexplored area of investigation. In order to understand how the two different types of subunits recognize glycine and glutamate, and to probe possible modes of subunit–subunit interaction, we have undertaken a crystallographic investigation of the NR1 S1S2 ligand-binding core. Ligand-binding specificity and affinity The crystal structures described here demonstrate that the mode of agonist binding to NR1 S1S2 involves two electrostatic interactions with residues on domain 1 and a series of hydrogen bonds with amino acids on domain 2: the α-carboxylate binds to the guanidinium group of Arg523, the α-amino moiety interacts with the carboxy group of Asp732, and the α-carboxylate and/or R-group forms hydrogen bonds to residues at the N-terminus of helix F on domain 2. Disruption of Arg523 abolishes the agonist response (Hirai et al., 1996) and the mutation of Asp732 to Asn or Glu shifts the glycine EC50 ∼14 500- and 4200-fold, respectively (Williams et al., 1996). These studies reinforce the importance of correctly positioned positive and negative charges in the binding pocket (Lampinen et al., 1998). Agonist binding is critically dependent upon a series of hydrogen bonds to side-chain and main-chain atoms, as well as to water molecules. Indeed, we suggest that DS binds more tightly to the receptor in comparison with glycine because it makes three additional hydrogen bonds and displaces a water molecule (W2). The selectivity of glycine over L-glutamate, or DS over L-serine, is very stringent; the affinity of L-glutamate to the NR1 subunit is so weak as to be unmeasurable (Miyazaki et al., 1999) and L-serine binds 300-fold less tightly than DS. Our NR1 S1S2 crystal structure, together with the GluR2 S1S2 glutamate structure (Armstrong and Gouaux, 2000), clearly explain the selectivity of glycine over glutamate and of DS over L-serine. The glycine selectivity is defined by the chemical environment and steric restraint created by primarily two amino acid side chains: Val689 and Trp731 preclude the binding of the L-glutamate γ-carboxyl group by removing a hydrogen bond donor and installing a steric barrier. D-isomer specificity is the consequence of steric constraints imposed by Phe484 and the hydrophobic environment created by Phe484 and Trp731. If one models L-serine into the binding pocket using the α-substituents of DS as a guide, then the hydroxyl group of L-serine unfavorably interacts with the phenyl ring of Phe484. By contrast, the hydroxyl group of DS is correctly positioned to form hydrogen bonds with Thr518, Asp732 and Ser688. The binding of the partial agonist DCS is similar to the binding of glycine and DS, and involves electrostatic interactions with the side chains of Arg523 and Asp732. However, the affinity of NR1 S1S2 for DCS is 34-fold lower than that for DS. To some extent, this difference in affinity can be explained by the fact that the pKa values of the key ionizable groups on DCS are closer to neutrality in comparison with the pKa values for the corresponding groups of DS. Indeed, the pKa values for the exocyclic oxygen and amino group of DCS are 4.5 and 7.5, respectively, whereas the values for the α-carboxy group and amino group of DS are 2.2 and 9.1, respectively (McBain et al., 1989). Therefore, the concentration of the doubly ionized and presumably tightly binding form of DCS is relatively lower in comparison with the corresponding form of DS. An additional difference is that DCS does not contain a hydroxyl group that can form a hydrogen bond with the carboxylate group of Asp732. The binding of DCKA is distinct from the binding of agonists in that DCKA is involved in π-stacking interactions with the aromatic ring of Phe484. Mutation of Phe484 severely disrupts the binding of 7-chlorokynurenic acid, an antagonist closely related to DCKA, to the NR1 receptor (Kuryatov et al., 1994). In GluR2 S1S2, the residue located at the same relative position as Phe484 is Tyr450 and in the GluR2 S1S2 5,6-dinitroquinoxalinedione (DNQX) cocrystal structure the quinoxaline ring of DNQX participates in π-stacking interactions with Tyr450 (Armstrong and Gouaux, 2000). π-stacking interactions are probably a common feature in the binding of aromatic antagonists to iGluRs. Kynurenic acid is less potent than the halogen-substituted antagonists, such as 7-chloro- and 5,7-dichlorokynurenic acid. In our DCKA structure, the 5- and 7-chloro substituents are ∼3.4 and 3.2 Å from the edges of the aromatic residues Trp731 and Phe408, respectively, and thus participate in weak but favorable hydrogen bonding and van der Waals interactions. The chloro substituents also lower the pKa of the aromatic ring substituents, which may enhance DCKA binding because the hydroxyl/carbonyl group is near the N-terminus of helix F, a known site for anion binding in both AMPA and NMDA receptors. Relationship between domain closure and receptor activation The crystallographic analysis of GluR2 S1S2 has revealed that the bilobed structure is ‘closed’ in the presence of agonist and ‘open’ in the apo state. A GluR2 antagonist, DNQX, stabilizes the ‘open’ apo-like conformation (Armstrong and Gouaux, 2000). The molecular movement resulting from the agonist-induced domain closure, in the context of the non-desensitized dimer, leads to separation of the regions proximal to the ion-channel gate and to activation or opening of the ion channel (Sun et al., 2002). Consistent with GluR2 S1S2, agonists and the antagonist DCKA stabilize NR1 S1S2 in ‘closed’ and ‘open’ conformations, respectively. The extent of domain closure in going from the antagonist- to agonist-bound states is greater in the case of NR1 S1S2 (∼21°) than in GluR2 S1S2 (∼16°). In terms of domain separation, the DCKA-bound NR1 S1S2 is more open than the DNQX-bound form of GluR2 S1S2 and is more closed than the apo state of GluR2 S1S2. We predict that the apo state of NR1 S1S2 will have a conformation that is similar to the DCKA-bound state. Furthermore, we suggest that the mechanism of NMDA receptor gating involves agonist-induced domain closure followed by the opening of the ion channel. The full and partial agonist structures of NR1 S1S2 clearly indicate that agonists stabilize hydrogen-bonding interactions between residues on domain 1 (Gln405) and domain 2 (Trp731 and Asp732) in the vicinity of the ligand-binding pocket. By contrast, the antagonist DCKA disrupts the interdomain hydrogen bonds and stabilizes an ‘open’-cleft conformation of the NR1 S1S2. Consistent with the above observation, the non-conservative substitution of Gln405 by Lys increases the glycine EC50 14 230-fold (Kuryatov et al., 1994). In GluR2 S1S2, there is an analogous interdomain interaction between Glu402 and Thr686 that stabilizes the activated closed-cleft conformation of the receptor (Armstrong et al., 1998; Armstrong and Gouaux, 2000). In the NR1 S1S2 structure, Ala714 occupies a position that is equivalent to Thr686 in GluR2 S1S2, and when Ala714 is mutated to leucine, the resulting receptor has an apparent reduced affinity for glycine but DCKA inhibition is unaffected (Wood et al., 1999). In the agonist/partial agonist-bound NR1 S1S2 structures, Ala714 is located at the N-terminus of helix I and is only 3.8 Å away from side chain of Gln405. We suggest that the Ala714 to leucine mutation destabilizes the glycine-bound closed-cleft conformation of NR1 S1S2, and therefore the effect of the mutation is greatest on full and partial agonists. The finding that the degree of domain closure for partial agon