Compilation of References on Conotoxin MVIIA Structure and Function (6 August 1996)
Biochemistry 34: 10256-10265 (1995)
Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin MVIIA.
T. Kohno, J. I. Kim, K. Kobayashi, Y. Kodera, T. Maeda & K. Sato
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
The three-dimensional solution structure of omega-conotoxin MVIIA, a 25-mer peptide antagonist of N-type calcium channels, was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 13 converged structures of omega-conotoxin MVIIA were obtained on the basis of 273 experimental constraints, including 232 distance constraints obtained from nuclear Overhauser effect (NOE) connectivities, 22 torsion angle (phi, chi 1) constraints, and 19 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.47 +/- 0.08 A for the backbone atoms (N, C alpha, C) and 1.27 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of omega-conotoxin MVIIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as that reported for omega-conotoxin GVIA, another N-type calcium channel blocker. The orientation of beta-stranded structure is similar to each other, suggesting that the conserved disulfide bond combination is essential for the molecular folding. We have recently determined by using alanine substitution analyses that Tyr 13 is essential for the activity of both toxins. On the basis of functional and structural analysis, it is shown that both omega-conotoxin MVIIA and GVIA retain a similar conformation to locate Tyr 13 in the appropriate position to allow binding to N-type calcium channels. These results provide a molecular basis for understanding the mechanism of calcium channel modulation through the toxin-channel interaction and insight into the discrimination of different subtypes of calcium channels.
Biochemistry 26: 2086-90 (1987)
Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom.
B. M. Olivera, L. J. Cruz, S. a. n. t. o. s. de, G. W. LeCheminant, D. Griffin, R. Zeikus, J. M. McIntosh, R. Galyean, J. Varga, W. R. Gray & ...
The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.
Biochem Biophys Res Commun 206: 449-454 (1995)
Tyr13 is essential for the activity of omega-conotoxin MVIIA and GVIA, specific N-type calcium channel blockers.
J. I. Kim, M. Takahashi, A. Ohtake, A. Wakamiya & K. Sato
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Two analogs of omega-conotoxin MVIIA, a 25mer peptide neurotoxin, were synthesized by replacing Lys2 or Tyr13 with Ala. The activities of synthetic analogs were estimated from the inhibitory action on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes. As in the case of omega-conotoxin GVIA, replacement of Tyr13 resulted in an enormous reduction in activity. In contrast, substitution of Ala for Lys2 gave only a small effect. These results indicate that Tyr13 is a critical amino acid of omega-conotoxin MVIIA and GVIA for blocking N-type calcium channel function.
FEBS Lett 370: 163-169 (1995)
Solution structure of omega-conotoxin MVIIA using 2D NMR spectroscopy.
V. J. Basus, L. Nadasdi, J. Ramachandran & G. P. Miljanich
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
The solution structure of omega-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 20-21, and 24-25. The structure of this toxin is very similar to that of omega-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.
J Mol Biol 234: 405-20 (1993)
Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin.
P. K. Pallaghy, B. M. Duggan, M. W. Pennington & R. S. Norton
NMR Laboratory, Biomolecular Research Institute, Parkville, Australia.
The 27 amino acid residue polypeptide omega-conotoxin GVIA, from venom of the cone shell Conus geographus, blocks neuronal voltage-activated calcium channels at picomolar concentrations. The three-dimensional structure in aqueous solution of synthetic omega-conotoxin has been determined from two-dimensional 1H n.m.r. data recorded at 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin-spin coupling constants were used as input for distance geometry calculations with the program DSPACE. The structures were then refined using back-calculation of NOESY spectra. The family of structures obtained in this way is well defined by the n.m.r. data, the best 12 structures having pairwise root-mean-square differences of 0.68 (+/- 0.15) A over the backbone heavy atoms (N, C alpha and C) and 1.15 (+/- 0.17) A over all heavy-atoms. The molecule adopts a compact structure consisting of a small, triple-stranded, anti-parallel beta-sheet and several reverse turns. All three tyrosine residues are located on the molecular surface, which is noteworthy for its abundance of side-chain hydroxyl groups. There is no negatively charged group in conotoxin, but the five positively charged groups are distributed in three small patches on the surface, one of which, made up of the ammonium moieties of the N terminus and Lys2, may contribute to the receptor-binding surface of the molecule. An isomer of conotoxin with the same amino acid sequence, but different disulfide pairings, has also been investigated. Its structure is less well ordered than that of native conotoxin and it shows significant heterogeneity, probably as a result of cis-trans isomerism preceding hydroxyproline residues.
J Biol Chem 262: 9877-82 (1987)
Identification of the receptor for omega-conotoxin in brain. Probable components of the calcium channel.
T. Abe & H. Saisu
Recently omega-conotoxin GVIA was shown to specifically block neuronal and other calcium channels. In this work, an azidonitrobenzoyl derivative of mono-[125I]iodo-omega-conotoxin GVIA was used to identify the components of its receptor site in synaptic plasma membrane by photoaffinity labeling. Components of Mr approximately equal to 310,000, approximately equal to 230,000, and 34,000 were specifically photolabeled. The characteristics of photolabeling of these three components were consistent with those of the specific binding of omega-conotoxin GVIA to synaptic plasma membrane with respect to the effects of metal ions, conventional calcium antagonists, and an agonist (1,4-dihydropyridines, verapamil, and diltiazem, etc.), omega-conotoxins GVIIA and GVIIB. Furthermore, the distribution of these three components in subcellular fractions from rat brain as estimated by photolabeling was in good agreement with that of the specific binding of omega-conotoxin GVIA to its receptor. These findings indicate that the components of Mr approximately equal to 310,000, approximately equal to 240,000, and 34,000 are the receptor for omega-conotoxin GVIA and suggest that these components are constituents of the voltage-sensitive calcium channel in brain. No specific photolabeling was observed in the plasma membrane of human erythrocytes, probably indicating the absence of the receptor for omega-conotoxin GVIA in the membrane.
FEBS Lett 235: 178-82 (1988)
Omega-conotoxin binding and effects on calcium channel function in human neuroblastoma and rat pheochromocytoma cell lines.
E. Sher, A. Pandiella & F. Clementi
Dept of Medical Pharmacology, University of Milan, Italy.
Binding of omega-conotoxin, a peptide toxin specific for some subtypes of voltage-operated calcium channels (VOCCs), was investigated in IMR32 neuroblastoma and PC12 pheochromocytoma cell lines. In both cell types, binding was specific, saturable and of high affinity. Association was rapid and dissociation almost non-existent. Dihydropyridines and verapamil failed to affect toxin binding, while high concentrations of CaCl2 completely antagonized it. Depolarization with high K+ induced a [Ca2+]i rise (revealed by the fura-2 fluorimetric technique) that consisted of an initial (0.5-1 min) peak followed by a prolonged (several minutes) plateau phase. omega-Conotoxin blocked mainly the first phase, while the dihydropyridine Ca2+ channel blocker, nitrendipine, primarily affected the plateau. This result suggests that in the two cell lines investigated, omega-conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.
J Bacteriol 175: 1235-8 (1993)
Inhibition of Escherichia coli chemotaxis by omega-conotoxin, a calcium ion channel blocker.
L. S. Tisa, B. M. Olivera & J. Adler
Department of Biochemistry, University of Wisconsin-Madison 53706-1569.
Escherichia coli chemotaxis was inhibited by omega-conotoxin, a calcium ion channel blocker. With Tris-EDTA-permeabilized cells, nanomolar levels of omega-conotoxin inhibited chemotaxis without loss of motility. Cells treated with omega-conotoxin swam with a smooth bias, i.e., tumbling was inhibited.
Int J Pept Protein Res 43: 363-366 (1994)
Synthesis of disulfide-bridged fragments of omega-conotoxins GVIA and MVIIA. Use of Npys as a protecting/activating group for cysteine in Fmoc syntheses.
R. G. Simmonds, D. E. Tupper & J. R. Harris
Lilly Research Centre Ltd., Eli Lilly & Co., Windlesham, Surrey, UK.
The 3-nitro-2-pyridinesulphenyl (Npys) moiety is finding increasing utility as a protecting-activating group for cysteine, particularly in the synthesis of cyclic and unsymmetrical disulfides using the Boc strategy. This chemistry has been extended to peptides assembled by the Fmoc strategy. N-Terminal Cys(Npys) is introduced via Boc-Cys(Npys)-OPfp. Non-N-terminal Cys(Npys) is incorporated by reacting a resin-bound, fully protected Cys(Acm) peptide with NpysCl. This approach has been applied to the synthesis of four disulfide-bridged fragments of omega-conotoxins GVIA and MVIIA.
Neuropharmacology 32: 1141-9 (1993)
A new Conus peptide ligand for Ca channel subtypes.
V. D. Monje, J. A. Haack, S. R. Naisbitt, G. Miljanich, J. Ramachandran, L. Nasdasdi, B. M. Olivera, D. R. Hillyard & W. R. Gray
Department of Biology, University of Utah, Salt Lake City 84112.
A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.
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