Structural dynamics of the glycine-binding domain of the N-Methyl-D-aspartate receptor

Abstract

N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycineand glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain ofNMDAreceptors showed no significant changes between the partial and full agonistbound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-ami-no-1-cyclobutanecarboxylic acid (ACBC) and L -alanine, and full agonists glycine and D -serine. For these studies, we have incorporated the unnatural amino acid p-acetyl- L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.