Characterization and three-dimensional structure determination of ψ-conotoxin PIIIF, a novel noncompetitive antagonist of nicotinic acetylcholine receptors

A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), phi-conotoxin PIIIF, was isolated from the venom of Conus purpurascens and found to have the sequence GOOCCLYGSCROFOGCYNALCCRK-NH2. The sequence is highly homologous to that of phi-conotoxin PIIIE, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both phi-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but phi-PIIIF is less potent by a factor of 10(1) - 10(2). A high-resolution structure of phi-PIIIF was determined by NMR and molecular modeling calculations. phi-PIIIF analogues containing [C-13]-labeled cysteine at selected positions were synthesized to resolve spectral overlap of Cys side chain proton signals. The structures are well-converged, with backbone atom position RMSDs of 0.21 Angstrom for the main body of the peptide between residues 4 and 22 and 0.47 Angstrom for all residues. The overall backbone conformation is closely similar to phi-PIIIE, the main difference being in the degree of conformational disorder at the two termini. phi-PIIIE and phi-PIIIF have similar locations of positive charge density, although phi-PIIIF has a lower overall charge. One disulfide bridge of phi-PIIIF appears to undergo dynamic conformational fluctuations based on both the model and on experimental observation. Chimeras in which the three intercysteine loops were swapped between phi-PIIIE and phi-PIIIF were tested for inhibitory activity against Torpedo nAChRs. The third loop, which contains no charged residues in either peptide, is the prime determinant of potency in these phi-conotoxins.