SOLUTION STRUCTURE OF OMEGA-CONOTOXIN MVIIC, A HIGH-AFFINITY LIGAND OF P-TYPE CALCIUM CHANNELS, USING H-1-NMR SPECTROSCOPY AND COMPLETE RELAXATION MATRIX ANALYSIS

We have determined the solution structure of the omega-conotoxin MVIIC from Conus magnus by H-1 NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-se nsitive Ca2+ channels in neurons. This 26 residue peptide with three d isulfide bonds was chemically synthesized and refolded for NMR structu ral studies. The H-1 NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the beta pro chiral centers. Complete relaxation matrix analysis using MARDIGRAS wa s used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined b y measuring C-13 relaxation times using inversely detected natural abu ndance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force field in vacuo. H-1 -H-1 vicinal coupling constants were obtained using a combination of l ine fitting of both E. COSY and NOESY spectra and used to generate ang le restraints that were included explicitly in the restrained molecula r dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 Angstrom. The ensemble R(1/6) factor calculated by CORMA for the final 15 structures was 11%. The final structure con sists of an anti-parallel, triple-stranded beta-sheet, with four turns . In spite of significant differences in amino acid sequence and affin ities for calcium channel subtypes, the backbone structure of omega-co notoxin MVIIC is very similar to the previously reported structure of omega-conotoxin GVIA.