Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha(Cys(192)-Cys(193)) at the alpha gamma interface of the nicotinicacetylcholine receptor

We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snak e neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) locat ed at the alpha gamma interface of the Torpedo nicotinic acetylcholine rece ptor. Each toxin position was substituted individually for a cysteine, whic h was then linked to a maleimido moiety through three different spacers, va rying in length from 10 to 22 Angstrom. We estimated the coupling efficienc y between the 15 toxin derivatives and the reduced cystine alpha(192-193) b y gel densitometry of Coomassie Blue-stained gels. A nearly quantitative co upling was observed between alpha Cys(192) and/or (alpha Cys(193) and all p robes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis cy-neurotoxin. These data sufficed to locate the reactive thiolate in a ''croissant-shaped" volume comprised between th e first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Alt ogether, the data suggest that (alpha Cys(192) and/or alpha Cys(193) at the alpha gamma interface of a muscular-type acetylcholine receptor, is tare) located in a volume located between 11.5 and 15.5 Angstrom from the alpha-c arbons at positions 10 and 33 of the toxin, under the tip of the toxin firs t loop and close to the second one.