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

J Biol Chem. 2015 Jan 9;290(2):797-804. doi: 10.1074/jbc.M114.605436. Epub 2014 Nov 17.

Abstract

N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound 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-amino-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.

Keywords: Fluorescence Resonance Energy Transfer (FRET); Glutamate Receptor; Ion Channel; Ionotropic Glutamate Receptor; LRET; Ligand-binding Protein; NMDA Receptor; Protein Dynamics; Receptor Structure-Function.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alanine / chemistry
  • Animals
  • Binding Sites
  • Crystallography, X-Ray
  • Fluorescence Resonance Energy Transfer
  • Glycine / chemistry*
  • Glycine / metabolism
  • Ligands
  • Models, Molecular
  • Protein Binding
  • Protein Conformation*
  • Protein Structure, Tertiary
  • Rats
  • Receptors, N-Methyl-D-Aspartate / agonists
  • Receptors, N-Methyl-D-Aspartate / chemistry*
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Structure-Activity Relationship

Substances

  • Ligands
  • Receptors, N-Methyl-D-Aspartate
  • Alanine
  • Glycine