Received for publication June 1, 2006.
Revised July 17, 2006.
Accepted for publication July 17, 2006.

Solution NMR of Acetylcholine Binding Protein Reveals ACh-mediated Conformational Change of the C-loop

Abstract

Previous x-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that following binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. Here we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with 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 TROSY ¹⁵N-¹H heteronuclear single quantum correlation (HSQC) spectroscopy of apo AChBP reveals 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. Following 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.

Key words: Nicotinic cholinergic, NMR

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