The solution structure of the complex formed between alpha-bungarotoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica

The region encompassing residues 181-98 on the alpha1 subunit of the muscle -type nicotinic acetylcholine receptor forms a major determinant for the bi nding of cu neurotoxins. We have prepared an N-15-enriched (18)-amino acid peptide corresponding to the sequence in this region to facilitate structur al elucidation by multidimensional NMR. Our aim was to determine the struct ural basis for the high affinity, stoichiometric complex formed between thi s cognate peptide and cu-bungarotoxin, a long cu-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experim ents, and the resulting interproton distance constraints were used to gener ate ensemble structures of the complex. Thr(8), Pro(10), Lys(38), Val(39), Val(40), and pro(69) in alpha -bungarotoxin and Tyr(189), Tyr(190), Thr(191 ), Cys(192), Asp(195), and Thr(196) in the peptide participate in major int ermolecular contacts. A comparison of the free and bound Lu-bungarotoxin st ructures reveals significant conformational rearrangements in flexible regi ons of alpha -bungarotoxin, mainly loops I, II, and the C-terminal tail. Fu rthermore, several of the calculated structures suggest that cation-pi inte ractions may be involved in binding. The root mean square deviation of the polypeptide backbone in the complex is 2.07 Angstrom. This structure provid es, to date, the highest resolution description of the contacts between a p rototypic alpha -neurotoxin and its cognate recognition sequence.