title: Structural model of the N-methyl-D-aspartate receptor glycine site probed by site-directed chemical coupling.
abstract: The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.
authors.affiliation: Laboratoire de Chimie Bioorganique, CNRS UMR 7514, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, F-67401 Illkirch, France. foucaud@bioorga.u-strasbg.fr
authors.forename: Bernard
authors.initials: B
authors.lastname: Foucaud
authors.forename: Bodo
authors.initials: B
authors.lastname: Laube
authors.forename: Rudolf
authors.initials: R
authors.lastname: Schemm
authors.forename: Annett
authors.initials: A
authors.lastname: Kreimeyer
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
authors.forename: Heinrich
authors.initials: H
authors.lastname: Betz
mesh_terms: D000818:Animals; D001665:Binding Sites; D004594:Electrophysiology; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry; D005998:Glycine; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes; D015202:Protein Engineering / Q000379:methods; D015363:Quinolones / Q000737:chemistry; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry*; D014981:Xenopus
chemical_list: D018691:Excitatory Amino Acid Antagonists; C409466:NR1 NMDA receptor; D015363:Quinolones; D016194:Receptors, N-Methyl-D-Aspartate; C094635:L 701324; D005998:Glycine