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...