STRUCTURAL ELEMENTS IN ALPHA-CONOTOXIN IMI ESSENTIAL FOR BINDING TO NEURONAL ALPHA(7) RECEPTORS

The neuronal-specific toxin a-conotoxin ImI (CTx ImI) has the sequence y-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cyst eine forms a disulfide bridge to produce a constrained two-loop struct ure. To investigate the structural basis for bioactivity we mutated in dividual residues in CTx ImI and determined bioactivity, Bioactivity o f the toxins was determined by their competition against I-125-labeled alpha-bungarotoxin binding to homomeric receptors containing alpha(7) sequence in the major extracellular domain and 5HT-3 sequence elsewhe re. The results reveal two regions in CTx ImI essential for binding to the alpha(7)/-5HT-3 receptor. The first is the triad Asp-Pro-Arg in t he first loop, where conservative mutations of each residue diminish a ffinity 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 over all results suggest that within the triad of the first loop, Pro posit ions 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.