Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinicacetylcholine receptor

The alpha -neurotoxins are three-fingered peptide toxins that bind selectiv ely at interfaces formed by the alpha subunit and its associating subunit p artner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. B ecause the alpha -neurotoxin from Naja mossambica mossambica I shows an unu sual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta se quences governing alpha -toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha -toxin. Analysis of the alpha gamma in terface shows unique pairwise interactions between the charged residues on the alpha -toxin and three regions on the alpha subunit located around resi due Asp(99), between residues Trp(149) and Val(153) and between residues Tr p(187) and Asp(200). Substitutions of cationic residues at positions betwee n Trp(149) and Val(153) markedly reduce the rate of alpha -toxin binding, a nd these cationic residues appear to be determinants in preventing alpha -t oxin binding to alpha2, alpha3, and alpha4 subunit containing receptors. Re placement of selected residues in the alpha -toxin shows that Ser(8) on loo p I and Arg(33) and Arg(36) on the face of loop II,in apposition to loop I, are critical to the alpha -toxin for association with the alpha subunit. P airwise mutant cycle analysis has enabled us to position residues on the co ncave face of the three alpha -toxin loops with respect to alpha and gamma subunit residues in the alpha -toxin binding site. Binding of NmmI alpha -t oxin to the alpha gamma interface appears to have dominant electrostatic in teractions not seen at the alpha delta interface.