Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2AN-Methyl-d-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling

Abstract

We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.The NMDA receptor channel is thought to be formed from the coassembly of two NR1 subunits and two NR2 subunits in a dimer of dimers configuration (Schorge and Colquhoun, 2003). NMDA receptors are unique among ligand-gated ion channels in that the binding of two different ligands is required for the activation of the receptor channel complex. Glycine, a coagonist, binds to residues located in the NR1 subunits, whereas glutamate binds to residues located in NR2 subunits (for review, see Erreger et al., 2004) of which there are four types (termed NR2A-D or ⑀ 1-4) and which are the major determinants of the pharmacological and biophysical properties of these receptors (Monyer et al., 1992(Monyer et al., , 1994Vicini et al., 1998;Wyllie et al., 1998). Ionotropic glutamate receptor subunits are comprised of distinct functional regions-an amino terminal domain, a ligand binding domain, a membrane-associated region, and an intracellular carboxyl-terminal domain (Fig. 1A). The ligand binding domain is thought to form a hinged clamshell-like structure (Armstrong et al., 1998) and is comprised of a region preceding the first membrane spanning domain (termed S1) and a region between the second and third membrane spanning domains (termed S2). In NR2 NMDA receptor s...

Disruption of Interdomain Interactions in the Glutamate Binding Pocket Affects Differentially Agonist Affinity and Efficacy of N-methyl-d-aspartate Receptor Activation

“…This model of the NR2A LBD with bound L-glutamate was generated based on the published (10) crystal structure of the GluR2 binding domain (Protein Data Bank entry 1FTJ) using the Sybyl 6.9 software (Tripos Associates) with residue numbering as in Ref. 24. The modeling procedure was analogous to that described previously for the binding domain of NR2B (25).…”

“…These values are based on a set of 1073 known protein structures and a distance cut-off of 4.5 Å used to define an interaction. (24) and moves toward the agonist and the D1 lobe during domain closure. Thus, D1D2 contacts in this region are induced by the agonist indirectly by promoting the movement of helix F.…”

Mechanism of Partial Agonism at NMDA Receptors for a Conformationally Restricted Glutamate Analog

“…A homology model of glutamate docked into the S1-S2 region of NR2A (GenBank accession number D13211) has been described previously (Chen et al, 2005). Glutamate was removed from the binding site, and homoquinolinate was built in its place, with the nitrogen of the aromatic ring left unprotonated.…”

“…Figure 2 A illustrates the alignment used to generate a homology model of the glutamate binding domain of NR2A (Chen et al, 2005) based on a crystal structure of the NR1 glycine binding domain (Furukawa and Gouaux, 2003). The ␣-helices and ␤-sheets were labeled according to the NR1 structure (Furukawa and Gouaux, 2003).…”

“…Figure 2 B-D superimposes the model of the NR2A S1S2 binding domain with either glutamate or homoquinolinate docked using MD. Figure 3A shows the glutamate ␣-carboxyl anchored by Arg499 plus a backbone nitrogen from Ser670 (Chen et al, 2005). Arg499 also makes an important hydrogen bond with the backbone carbonyl of Thr494 (data not shown).…”