SOLUTION STRUCTURES OF A HIGHLY INSECTICIDAL RECOMBINANT SCORPION ALPHA-TOXIN AND A MUTANT WITH INCREASED ACTIVITY

The solution structure of a recombinant active alpha-neurotoxin from L eiurus quinquestriatus hebraeus, Lqh alpha IT, was determined by proto n two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Th is toxin is the most insecticidal among scorpion cl-neurotoxins and, t herefore, serves as a model for clarifying the structural basis for th eir biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 Angstrom. T hese structures had root mean square deviations of 0.49 and 1.00 Angst rom with respect to the average structure for backbone atoms and all h eavy atoms, respectively. Similarly to other scorpion toxins, the stru cture of Lqh alpha IT consists of an alpha-helix, a three-strand antip arallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a ''herringbone'' arrangement. Positive phi angles were found for Ala(50 ) and Asn(11), suggesting their proximity to functionally important re gions of the molecule. The sample exhibited conformational heterogenei ty over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh alpha IT is ver y similar to that of the most active antimammalian scorpion alpha-toxi n, AaHII, from Androctonus australis Hector (60% amino acid sequence h omology). Yet, several important differences were observed at the 5-re sidue turn comprising residues Lys(8)-Cys(12), the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of th e R64H mutant, which is 3 times more toxic than the unmodified Lqh alp ha IT, demonstrated the importance of the spatial orientation of the l ast residue side chain for toxicity of Lqh alpha IT.