title: Characterization and three-dimensional structure determination of psi-conotoxin Piiif, a novel noncompetitive antagonist of nicotinic acetylcholine receptors.
abstract: A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), psi-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 psi-conotoxin Piiie, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both psi-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but psi-Piiif is less potent by a factor of 10(1)-10(2). A high-resolution structure of psi-Piiif was determined by NMR and molecular modeling calculations. Psi-Piiif analogues containing [(13)C]-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 A for the main body of the peptide between residues 4 and 22 and 0.47 A for all residues. The overall backbone conformation is closely similar to psi-Piiie, the main difference being in the degree of conformational disorder at the two termini. Psi-Piiie and psi-Piiif have similar locations of positive charge density, although psi-Piiif has a lower overall charge. One disulfide bridge of psi-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 psi-Piiie and psi-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 psi-conotoxins.
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Van Wagoner
authors.forename: Richard B
authors.initials: RB
authors.lastname: Jacobsen
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Chris M
authors.initials: CM
authors.lastname: Ireland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D004594:Electrophysiology; D020128:Inhibitory Concentration 50; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008974:Mollusca; D018733:Nicotinic Antagonists / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013237:Stereoisomerism; D014101:Torpedo; D014982:Xenopus laevis / Q000378:metabolism; D020960:omega-Conotoxins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004220:Disulfides; D018733:Nicotinic Antagonists; D010455:Peptides; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D020960:omega-Conotoxins; C107492:psi-conotoxin PIIIE; C475537:psi-conotoxin PIIIF