Quiram_1998_J.Biol.Chem_273_11007

The neuronal-specific toxin alpha-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. To investigate the structural basis for bioactivity we mutated individual residues in CTx ImI and determined bioactivity. Bioactivity of the toxins was determined by their competition against 125I-labeled alpha-bungarotoxin binding to homomeric receptors containing alpha7 sequence in the major extracellular domain and 5HT-3 sequence elsewhere. The results reveal two regions in CTx ImI essential for binding to the alpha7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in the first loop, where conservative mutations of each residue diminish affinity by 2-3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring.