Structure-activity relationships in a peptidic alpha 7 nicotinic acetylcholine receptor antagonist

alpha -Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (n ACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha -conotoxin ImI, a 12 amino acid peptide active at a lpha7 nACh receptors, in order to gain insight into the primary and tertiar y structural requirements of neuronal alpha -conotoxin specificity. NMR sol ution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD val ues of 0.46, 0.2, and 0.62 Angstrom from their mean structures, respectivel y, for the backbone atoms N, C-alpha, and C' of residues 2-11. The R11E mut ant was found to have activity near that of wild-type ImI, while R7L and D5 N demonstrated activities reduced by at least two orders of magnitude. Comp arison of the structures reveals a common two-loop architecture, with varia tions observed in backbone and sidechain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures an d activities with those from previously published studies emphasizes that e xisting hypotheses regard -ing the molecular determinants of alpha -conotox in specificity are not adequate for explaining peptide activity, and sugges ts that more subtle features, visualized here at the atomic level, are impo rtant for receptor binding. These data, in conjunction with reported charac terizations of the acetylcholine binding site, support a model of toxin act ivity in which a single solvent-accessible toxin side-chain anchors the com plex, with supporting weak interactions determining both the efficacy and t he subtype specificity of the inhibitory activity. (C) 2000 Academic Press.