Three-dimensional Structure in Solution of the Calcium Channel Blocker ω-Conotoxin

Abstract

The 27 amino acid residue polypeptide ω-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 ω-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) Å over the backbone heavy atoms (N, C α and C) and 1·15(±0middot;17) Å over all heavy-atoms. The molecule adopts a compact structure consisting of a small, triple-stranded, anti-parallel β-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.