3-DIMENSIONAL STRUCTURE OF TOXIN OSK1 FROM ORTHOCHIRUS-SCROBICULOSUS SCORPION-VENOM

A 600 MHz H-1 NMR study of toxin OSK1, blocker of small-conductance Ca 2+-activated K+ channels, is presented. The unambiguous sequential ass ignment of all the protons of the toxin was obtained using TOCSY, DQF- COSY, and NOESY experiments at pH 3.0 (10, 30, and 45 degrees C) in aq ueous solution. (3)J(N alpha), (3)J(alpha beta) vicinal spin coupling constants were determined in high-resolution spectra. The cross-peak v olumes in NOESY spectra and the coupling constants were used to define the local structure of the protein by the program HABAS and to genera te torsion angle and interproton distance constraints for the program DIANA. Hydrogen-deuterium exchange rates of amide protons showed possi ble locations of hydrogen bonds. The hydrogen bond accepters and disul fide bridges between residues 8-28, 14-33, and 18-35 were determined w hen analyzing distance distribution in preliminary DIANA structures. A ll constraints were used to obtain a set of 30 structures by DIANA. Th e resulting rms deviations over 30 structures are 1.30 Angstrom for th e heavy atoms and 0.42 Angstrom for the backbone heavy atoms. The stru ctures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the alpha-helix (r esidues 10-21) slightly distorted at the Cys14 residue, two main stran ds of the antiparallel beta-sheet (24-29, 32-38), and the extended fra gment (2-6). The motif is stabilized by the disulfide bridges in the w ay, common to all known scorpion toxins. Using the fine spatial toxin structure, alignment of the homologues, mutagenesis analysis, and comp arison of scorpion toxin family functions, we delineate some differenc es significant for the toxin specificity.