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Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSαβ scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion a-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion β-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhαIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNav1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNav1 activation. Thus, the short CSαβ scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNav1. [ABSTRACT FROM AUTHOR]

The bioactive surface of scorpion β-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved βαββ core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian β-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated β-toxins (ΔΔBj-xtrIT and ΔΔCss4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, ΔΔBj-xtrIT and ΔΔCss4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion α-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion α-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of LqhαIT at insect sodium channels. Co-application of ΔΔBj-xtrIT or ΔΔCss4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at ΔΔBj-xtrIT and ΔΔCss4 capable of interaction with sodium channels at a site other than sites 3,4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both β-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold. [ABSTRACT FROM AUTHOR]

The affinity of scorpion α-toxins for various voltage-gated sodium channels (Navs) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8–12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Navs. We examined this hypothesis using Lqh3, an α-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Navs compared with other α-toxins and its binding to insect Navs is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other α-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9–10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Navs expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling α-like toxins to affect a broad range of Navs. [ABSTRACT FROM AUTHOR]

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232–235). This suggestion was adopted by Smith et al. (Smith, J. I., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127–11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion β-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion β-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNav1 and rat brain rNav1.2a develops. We show that in general, scorpion β-toxins do not partition in neuronal membranes and that in the case in which a depressant β-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of E-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion β-toxins is accessible to the extracellular solvent. [ABSTRACT FROM AUTHOR]

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Navs) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Nav1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Nays varies. [ABSTRACT FROM AUTHOR]

Scorpion β-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) 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 β-toxin, Css4, whose effects on rat brain Nays 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 Ų composed of the following: 1) a cluster of residues associated with the α-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion β-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the β2 and β3 strands, which is likely to confer the specificity for mammalian Navs; 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 β-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels. [ABSTRACT FROM AUTHOR]

Scorpion β-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 β-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 α-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" (Tyr26and Val34). Comparison of the Bj-xtrIT structure with that of other fi-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 β-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. [ABSTRACT FROM AUTHOR]

Scorpion toxins that affect sodium channel (NaCh) gating in excitable cells are divided into α- and β-classes. Whereas α-toxins have been found in scorpions throughout the world, anti-mammalian β-toxins have been assigned, thus far, to ‘New World’ scorpions while anti-insect selective β-toxins (depressant and excitatory) have been described only in the ‘Old World’. This distribution suggested that diversification of β-toxins into distinct pharmacological groups occurred after the separation of the continents, 150 million years ago. We have characterized a unique toxin, Lqhβ1, from the ‘Old World’ scorpion, Leiurus quinquestriatus hebraeus , that resembles in sequence and activity both ‘New World’β-toxins as well as ‘Old World’ depressant toxins. Lqhβ1 competes, with apparent high affinity, with anti-insect and anti-mammalian β-toxins for binding to cockroach and rat brain synaptosomes, respectively. Surprisingly, Lqhβ1 also competes with an anti-mammalian α-toxin on binding to rat brain NaChs. Analysis of Lqhβ1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying β-toxin activity. Moreover, Lqhβ1 resembles β-toxins by having a weak effect on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs. These multifaceted features suggest that Lqhβ1 may represent an ancestral β-toxin group in ‘Old World’ scorpions that gave rise, after the separation of the continents, to depressant toxins in ‘Old World’ scorpions and to various β-toxin subgroups in ‘New World’ scorpions. [ABSTRACT FROM AUTHOR]

Abstract: Scorpion depressant β-toxins show high preference for insect voltage-gated sodium channels (Navs) 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 Navs. 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. [Copyright &y& Elsevier]

The scorpion α-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Navs) and is inactive at insect Navs. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNav1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion α-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhαIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe15 of the core domain in toxin potency at rNav1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion α-toxins we were able to eliminate the activity of Lqh2 at rNav1.4 (skeletal muscle), hNav1.5 (cardiac), and rNav1.6 channels, with no hindrance of its activity at Nav1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent. [ABSTRACT FROM AUTHOR]

Scorpion α-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 α-toxin LqhαIT (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 α-toxin, Aah2 (from Androctonus australis hector). The chimera harboring both domains, Aah2LqhαIT(face), was as active to insects as LqhαIT. Structure determination of Aah2LqhαIT(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 LqhαIT. Notably, such a protrusion is observed in all α-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 α-toxin specificity for insects and suggest a putative binding mechanism to insect NaChs. [ABSTRACT FROM AUTHOR]