Solution Structure of ω-Conotoxin MVIIC, a High Affinity Ligand of P-type Calcium Channels, using 1H NMR Spectroscopy and Complete Relaxation Matrix Analysis

We have determined the solution structure of the ω-conotoxin MVIIC from Conus magusby 1H NMR. This conopeptide preferentially blocks P and Q type Ca 2+currents by binding with high affinity to voltage-sensitive Ca 2+channels in neurons. This 26 residue peptide with three disulfide bonds was chemical...

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Bibliographic Details
Published in:Journal of molecular biology 1995-04, Vol.248 (1), p.106-124
Main Authors: Farr-Jones, Shauna, Miljanich, George P., Nadasdi, Laszlo, Ramachandran, J., Basus, Vladimir J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Calcium Channel Blockers - chemistry
Calcium Channels - metabolism
complete relaxation matrix analysis
Conus magus
Hydrogen
Hydrogen Bonding
Magnetic Resonance Spectroscopy - methods
Models, Molecular
Molecular Sequence Data
Mollusca
Mollusk Venoms - chemistry
neurotoxins
NMR structure
omega-Conotoxin GVIA
omega-Conotoxins
P-type calcium channels
Peptides - chemistry
Peptides - isolation & purification
Peptides - metabolism
Protein Conformation
Sequence Homology, Amino Acid
Solutions
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Summary:We have determined the solution structure of the ω-conotoxin MVIIC from Conus magusby 1H NMR. This conopeptide preferentially blocks P and Q type Ca 2+currents by binding with high affinity to voltage-sensitive Ca 2+channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the β prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance 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. 1H- 1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 Å. The ensemble R 1/6factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded β-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of ω-conotoxin MVIIC is very similar to the previously reported structure of ω-conotoxin GVIA.
ISSN:0022-2836
1089-8638
DOI:10.1006/jmbi.1995.0205