3-DIMENSIONAL SOLUTION STRUCTURE OF THE CALCIUM-CHANNEL ANTAGONIST OMEGA-AGATOXIN-IVA - CONSENSUS MOLECULAR FOLDING OF CALCIUM-CHANNEL BLOCKERS

The three-dimensional solution structure of omega-agatoxin IVA, which is a specific blocker of the P-type calcium channel isolated from funn el web spider venom and has a molecular mass of 5.2 kDa, was determine d by two dimensional H-1 NMR spectroscopy, combined with simulated ann ealing calculations. On the basis of 563 experimental constraints, inc luding 516 distance constraints obtained from the nuclear Overhauser e ffect, 21 torsion angle (phi,chi(1)) constraints, and 26 constraints a ssociated with hydrogen bonds and disulfide bonds, a total of 14 conve rged structures were obtained. The atomic root mean square difference for the 14 converged structures with respect to the mean coordinates i s 0.42 (+/-0.07) Angstrom for the backbone atoms (N, C-alpha, C) and 0 .95 (+/-0.15) Angstrom for all heavy atoms of the central part (residu es 4 to 38) constrained by four disulfide bonds. The N- and C-terminal segments (residues 1 to 3 and 39 to 48, respectively) have a disorder ed structure in aqueous solution. The molecular structure of omega-aga toxin IVA is composed of a short triple-stranded antiparallel beta-she et, three loops, and the disordered N- and C-terminal segments. The ov erall beta-sheet topology is +2x, -1, which is the same as that report ed for omega-conotoxin GVIA, an N-type calcium channel blocker. Irresp ective of differences in the number of disulfide bonds and low primary sequence homology, these two peptide toxins show a significant struct ural similarity in three dimensions. The whole-cell voltage-clamp reco rding using rat cerebellar slices suggests that the hydrophobic C-term inal segment of omega-agatoxin IVA, which does not exist in omega-cono toxin GVIA, plays a crucial role in the blocking action of omega-agato xin IVA on the P-type calcium channel in rat cerebellar Purkinje cells . The present study provides a molecular basis for the toxin-channel i nteraction, and thereby provides insight into the discrimination of di fferent subtypes of calcium channels. (C) 1995 Academic Press Limited