title: Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross-linkers.
abstract: Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.
authors.affiliation: Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai, China.|Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Paris, France.|Institut National de la Santé et de la Recherche Médicale, U1024, Paris, France.|Centre National de la Recherche Scientifique, UMR 8197, Paris, France.
authors.forename: Meilin
authors.initials: M
authors.lastname: Tian
authors.affiliation: Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Paris, France.|Institut National de la Santé et de la Recherche Médicale, U1024, Paris, France.|Centre National de la Recherche Scientifique, UMR 8197, Paris, France.
authors.forename: Shixin
authors.initials: S
authors.lastname: Ye
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000596:Amino Acids / Q000235:genetics* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017470:Receptors, Glutamate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D015032:Zinc / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D008024:Ligands; C121001:NR2B NMDA receptor; D010880:Piperidines; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D029867:Xenopus Proteins; C512833:nodal1 protein, Xenopus; D000255:Adenosine Triphosphate; D015032:Zinc; C010739:ifenprodil