Characterization and Three-Dimensional Structure Determination of ψ-Conotoxin Piiif, a Novel Noncompetitive Antagonist of Nicotinic Acetylcholine Receptors

• Published in: Biochemistry (Easton) Vol. 42; no. 21; pp. 6353 - 6362
• Main Authors: Van Wagoner, Ryan M, Jacobsen, Richard B, Olivera, Baldomero M, Ireland, Chris M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.06.2003
• Subjects: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Conus purpurascens; Disulfides; Electrophysiology; Inhibitory Concentration 50; Magnetic Resonance Spectroscopy; Marine; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Mollusca; Nicotinic Antagonists - chemistry; omega-Conotoxins - chemistry; omega-Conotoxins - metabolism; Oocytes - metabolism; Peptide Biosynthesis; Peptides - chemistry; Protein Conformation; Protein Folding; Protein Structure, Tertiary; psi -Conotoxin PIIIF; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - metabolism; Recombinant Fusion Proteins - chemistry; Stereoisomerism; Torpedo; Xenopus laevis - metabolism
• DOI: 10.1021/bi0272757
• ISSN: 0006-2960

A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), ψ-conotoxin Piiif, was isolated from the venom of Conus purpurascens and found to have the sequence GOOCCLYGSCROFOGCYNALCCRK-nh 2. The sequence is highly homologous to that of ψ-conotoxin Piiie, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both ψ-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but ψ-Piiif is less potent by a factor of 101−102. A high-resolution structure of ψ-Piiif was determined by NMR and molecular modeling calculations. ψ-Piiif analogues containing [13C]-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 Å for the main body of the peptide between residues 4 and 22 and 0.47 Å for all residues. The overall backbone conformation is closely similar to ψ-Piiie, the main difference being in the degree of conformational disorder at the two termini. ψ-Piiie and ψ-Piiif have similar locations of positive charge density, although ψ-Piiif has a lower overall charge. One disulfide bridge of ψ-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 ψ-Piiie and ψ-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 ψ-conotoxins.