X-ray Structure and Mutagenesis of the Scorpion Depressant Toxin LqhIT2 Reveals Key Determinants Crucial for Activity and Anti-Insect Selectivity Izhar Karbat 1 , Michael Turkov 1 , Lior Cohen 1 , Roy Kahn 1 , Dalia Gordon 1 Michael Gurevitz 1 ⁎ and Felix Frolow 2 ⁎ 1 Department of Plant Sciences, George S. Wise Faculty of Life Sciences, and The Daniella Rich Institute for Structural Biology , Tel-Aviv University , Tel-Aviv 69978, Israel 2 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, and The Daniella Rich Institute for Structural Biology , Tel-Aviv University , Tel-Aviv 69978, Israel Scorpion depressant β-toxins show high preference for insect voltage-gated sodium channels (Na v s) and modulate their activation. Although their pharmacological and physiological effects were described, their three- dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatus hebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 Å resolution. The toxin molecule is composed of a conserved cysteine-stabilized α/β-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na v s. The N-groove is flanked by Glu24 and Tyr28, which belong to the “pharmacophore” of scorpion β-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in “voltage-sensor trapping”, the mode of action that typifies scorpion β-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position. © 2006 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: scorpion depressant toxin; anti-insect selectivity; X-ray structure; bioactive surface Introduction Scorpion depressant toxins are 61 amino acid long polypeptides that show high preference for insect voltage-gated sodium channels (Na v s). The name of these toxins is derived from the paralytic symptoms they induce when injected to Sarcophaga falculata (blowfly) larvae, and the sustained mem- brane depolarization leading to block in evoked action potentials and loss of muscle tonus. 1–3 Depressant toxins were classified to the β-class of “long-chain” scorpion toxins due to their ability to modulate Na v activation, and to compete with excitatory scorpion toxins on binding to receptor- site 4 in insect neuronal membranes. 4,5 Yet, depres- sant toxins do not compete with anti-mammalian scorpion β-toxins on binding to rat neuronal membrane preparations, and are harmless when injected subcutaneously to mice. 6,7 Although a number of depressant toxins were identified and characterized (e.g. LqhIT 2 2,8 ; BjIT 2 1 ; BaIT 2 9,10 ; BotIT 4 , BotIT 5 11 ; LqqIT 2 12,13 ; AaIT 5 14,15 ), only a few were cloned 16 and very little is known about residues important for their activity. 3 In addition, no data about the three-dimensional structure of these toxins were reported. Abbreviations used: Na v , voltage-gated sodium channels; Dm, Drosophila melanogaster. E-mail addresses of the corresponding authors: mamgur@post.tau.ac.il; mbfrolow@post.tau.ac.il doi:10.1016/j.jmb.2006.10.085 J. Mol. Biol. (2007) 366, 586–601 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.