The α-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the α subunit and its associating subunit partner, γ, δ, or ε of the nicotinic acetylcholine receptor. Because the α-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the αγ and αδ over the αε subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the γ and δ sequences governing α-toxin recognition. To complement this approach, we have similarly analyzed residues on the α subunit face of the binding site dictating specificity for α-toxin. Analysis of the αγ interface shows unique pairwise interactions between the charged residues on the α-toxin and three regions on the α subunit located around residue Asp99, between residues Trp149 and Val153, and between residues Trp187 and Asp200. Substitutions of cationic residues at positions between Trp149 and Val153 markedly reduce the rate of α-toxin binding, and these cationic residues appear to be determinants in preventing α-toxin binding to α2, α3, and α4 subunit containing receptors. Replacement of selected residues in the α-toxin shows that Ser8 on loop I and Arg33 and Arg36 on the face of loop II, in apposition to loop I, are critical to the α-toxin for association with the α subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three α-toxin loops with respect to α and γ subunit residues in the α-toxin binding site. Binding of NmmI α-toxin to the αγ interface appears to have dominant electrostatic interactions not seen at the αδ interface.
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