NMR SEQUENTIAL ASSIGNMENTS AND SOLUTION STRUCTURE OF CHLOROTOXIN, A SMALL SCORPION TOXIN THAT BLOCKS CHLORIDE CHANNELS

The solution structure of chlorotoxin, a small toxin purified from the venom of the Leiurus quinquestriatus scorpion, has been determined us ing 2D H-1 NMR spectroscopy. Analysis of the NMR data shows that the s tructure consists of a small three-stranded antiparallel beta-sheet pa cked against an alpha-helix, thereby adopting the same fold as charybd otoxin and other members of the short scorpion toxin family [Arseniev et al. (1984) FEES Lett. 165, 57-62; Martins et al. (1990) FEES Lett. 260, 249-253; Bontems et al. (1991) Science 254, 1521-1523]. Three dis ulfide bonds of chlorotoxin (Cys5-Cys28, Cys16-Cys33, and Cys20-Cys35) , cross-linking the alpha-helix to the beta-sheet, follow the common p attern found in the other short scorpion toxins. The fourth disulfide bridge (Cys2-Cys19) links the small N-terninal beta strand to the rest of the molecule, in contrast to charybdotoxin where this disulfide br idge is absent and the first strand interacts with the rest of the mol ecule by several contacts between hydrophobic residues. Another struct ural difference between chlorotoxin and charybdotoxin is observed at t he level of the alpha-beta turn. This difference is accompanied by a c hange in the electrostatic potential surface, which is largely positiv e at the level of this turn in chlorotoxin, whereas no such positive p otential surface can be found at the same position in charybdotoxin. I n the latter protein, the positive surface is formed by different char ged residues situated on the solvent-exposed site of the C-terminal be ta-sheet. This positive region is disrupted in chlorotoxin by the muta tion of one of the arginines into a leucine and by the presence of two aspartic acid residues on the alpha-helix.