Scorpion ␣-toxins are similar in their mode of action and three-dimensional structure but differ considerably in affinity for various voltage-gated sodium channels (NaChs). To clarify the molecular basis of the high potency of the ␣-toxin Lqh␣IT (from Leiurus quinquestriatus hebraeus) for insect NaChs, we identified by mutagenesis the key residues important for activity. We have found that the functional surface is composed of two distinct domains: a conserved "Core-domain" formed by residues of the loops connecting the secondary structure elements of the molecule core and a variable "NC-domain" formed by a five-residue turn (residues 8 -12) and a C-terminal segment (residues 56 -64). We further analyzed the role of these domains in toxin activity on insects by their stepwise construction onto the scaffold of the anti-mammalian ␣-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2Lqh␣IT(face) , was as active to insects as Lqh␣IT. Structure determination of Aah2Lqh␣IT(face) by x-ray crystallography revealed that the NC-domain deviates from that of Aah2 and forms an extended protrusion off the molecule core as appears in Lqh␣IT. Notably, such a protrusion is observed in all ␣-toxins active on insects. Altogether, the division of the functional surface into two domains and the unique configuration of the NC-domain illuminate the molecular basis of ␣-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs.Voltage-gated sodium channels (NaChs) 1 mediate the transient increase in sodium ion permeability that triggers action potentials in excitable cells (1). These channels are composed of a pore-forming ␣-subunit (260 kDa) associated with one or two auxiliary ␤-subunits. The ␣-subunit consists of four repeat domains (D1-D4), each containing six transmembrane segments (S1-S6) and a membrane-associated re-entrant segment (SS1-SS2), connected by internal and external loops. A key feature in the function of NaChs is their gating behavior, namely the ability to rapidly activate and inactivate upon cell membrane depolarization, leading to transient increase in Na ϩ conductance (1). Due to their key role in excitability, these channels are targeted by a variety of toxins.Long-chain scorpion toxins are 61-to 76-residue-long polypeptides that share a similar core composed of an ␣-helix packed against a three-stranded ␤-sheet and stabilized by four disulfide bonds. These toxins bind to various receptor sites on the extracellular face of NaChs and alter their gating. Traditionally, they are divided into two major classes, ␣-and ␤-toxins, according to their mode of action and binding properties to distinct receptor sites on NaChs (2, 3).Scorpion ␣-toxins prolong the action potential by slowing channel inactivation, possibly through interference with the outward movement of the D4S4 segment necessary for the fast inactivation process (4). The scorpion ␣-toxin binding site, termed neurotoxin receptor site-3, has been shown to involve the extracellular regions of D...

Scorpion ␤-toxins that affect the activation of mammalian voltage-gated sodium channels (Na v s) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion ␤-toxin, Css4, whose effects on rat brain Na v s have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 Å 2 composed of the following: 1) a cluster of residues associated with the ␣-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion ␤-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the ␤2 and ␤3 strands, which is likely to confer the specificity for mammalian Na v s; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion ␤-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile 73 and Ile 74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

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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 Å 2 , 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, Glu 30 is a center of a putative "hot spot" in the toxin-receptor binding-interface and is shielded from bulk solvent by a hydrophobic "gasket" (Tyr 26 and Val 34 ). 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.

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

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