Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop

Mol Pharmacol. 2006 Oct;70(4):1230-5. doi: 10.1124/mol.106.027185. Epub 2006 Jul 17.

Abstract

Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylcholine / metabolism*
  • Amino Acid Substitution
  • Cell Line
  • Humans
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Molecular
  • Protein Conformation*
  • Protein Structure, Tertiary
  • Receptors, Cholinergic / chemistry
  • Solvents

Substances

  • Receptors, Cholinergic
  • Solvents
  • Acetylcholine