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 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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The structure of bioactive surfaces of proteins is a subject of intensive research, yet the mechanisms by which such surfaces have evolved are largely unknown. Polypeptide toxins produced by venomous animals such as sea anemones, cone snails, scorpions, and snakes show multiple routes for active site diversification, each maintaining a typical conserved scaffold. Comparative analysis of an array of genetically related scorpion polypeptide toxins that modulate sodium channels in neuronal membranes suggests a unique route of toxic site diversification. This premise is based on recent identification of bioactive surfaces of toxin representative of three distinct pharmacological groups and a comparison of their 3-dimensional structures. Despite their similar scaffold, the bioactive surfaces of the various toxins vary considerably, but always coincide with the molecular exterior onto which the C-tail is anchored. Superposition of the toxin structures indicates that the C-tails diverge from a common structural start point, which suggests that the pharmacological versatility displayed by these toxins might have been achieved along evolution via structural reconfiguration of the C-tail, leading to reshaping of new bioactive surfaces.