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J. Biol. Chem., Vol. 279, Issue 9, 8206-8211, February 27, 2004

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Dissection of the Functional Surface of an Anti-insect Excitatory Toxin Illuminates a Putative "Hot Spot" Common to All Scorpion -Toxins Affecting Na⁺ Channels^*

Lior Cohen, Izhar Karbat, Nicolas Gilles¶, Oren Froy||, Gerardo Corzo¶, Ruthie Angelovici, Dalia Gordon**, and Michael Gurevitz**

From the Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel, ^¶Commissariat à l'Energie Atomique, Départment d'Ingéniérie et d'Etudes des Protéines, C.E. Saclay F-91191, France, and ^||Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environmental Quality, Hebrew University of Jerusalem, Rehovot 76100, Israel

Scorpion -toxins affect the activation of voltage-sensitive sodium channels (NaChs). Although these toxins have been instrumental in the study of channel gating and architecture, little is known about their active sites. By using an efficient system for the production of recombinant toxins, we analyzed by point mutagenesis the entire surface of the -toxin, Bj-xtrIT, an anti-insect selective excitatory toxin from the scorpion Buthotus judaicus. Each toxin mutant was purified and analyzed using toxicity and binding assays, as well as by circular dichroism spectroscopy to discern the differences among mutations that caused structural changes and those that specifically affected bioactivity. This analysis highlighted a functional discontinuous surface of 1405 Ų, which was composed of a number of non-polar and three charged amino acids clustered around the main -helical motif and the C-tail. Among the charged residues, Glu³⁰ is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr²⁶ and Val³⁴). Comparison of the Bj-xtrIT structure with that of other -toxins that are active on mammals suggests that the hot spot and an adjacent non-polar region are spatially conserved. These results highlight for the first time structural elements that constitute a putative "pharmacophore" involved in the interaction of -toxins with receptor site-4 on NaChs. Furthermore, the unique structure of the C-terminal region most likely determines the specificity of excitatory toxins for insect NaChs.

Received for publication, July 14, 2003 , and in revised form, December 11, 2003.

^* This research was supported in part by United States-Israel Binational Agricultural Research and Development Grant IS-3259-01 (to D. G. and M. G.) and by Israeli Science Foundation Grants 508/00 (to D. G.) and 733/01 (to M. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed. E-mail: mamgur{at}post.tau.ac.il and dgordon{at}post.tau.ac.il.

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