The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica

Abstract

The region encompassing residues 181-98 on the α1 subunit of the muscle-type nicotinic acetylcholine receptor forms a major determinant for the binding of α-neurotoxins. We have prepared an 15N-enriched 18-amino acid peptide corresponding to the sequence in this region to facilitate structural elucidation by multi-dimensional NMR. Our aim was to determine the structural basis for the high affinity, stoichiometric complex formed between this cognate peptide and α-bungarotoxin, a long α-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experiments, and the resulting interproton distance constraints were used to generate ensemble structures of the complex. Thr8, Pro10, Lys38, Val39, Val 40, and Pro69 in α-bungarotoxin and Tyr 189, Tyr190, Thr191, Cys192, Asp195, and Thr196 in the peptide participate in major intermolecular contacts. A comparison of the free and bound α -bungarotoxin structures reveals significant conformational rearrangements in flexible regions of α-bungarotoxin, mainly loops I, II, and the C-terminal tail. Furthermore, several of the calculated structures suggest that cation-π interactions may be involved in binding. The root mean square deviation of the polypeptide backbone in the complex is 2.07 Å. This structure provides, to date, the highest resolution description of the contacts between a prototypic α-neurotoxin and its cognate recognition sequence.