Michael Gurevitz

Functional Anatomy of Scorpion Toxins Affecting Sodium Channels

AbstractLong chain scorpion toxins (made of 60 to 70 amino acids) acting on voltage-gated sodium channels in excitable cells are responsible for human envenomation, and comprise a-toxins that inhibit sodium current inactivation and p-toxins, that modify the activation process. These toxins may be further divided according to their pharmacological activities. Thus, a-toxins highly active on mammals or insects, as well as α like toxins may be distinguished within the α-toxin class. The β-toxin class includes toxins active on mammals and, as a separate group, the excitatory and depressant toxins active exclusively on insects. All these toxins possess 4 disulfide bridges and share 15 similar non cystine residues. Accordingly, their 3D structure is highly conserved, comprising an α-helix and a triple stranded β-sheet. The most solvent exposed turns of this structure are prone to insertions or deletions, and accordingly correspond to the most structurally variable regions of the toxins. They have been tested by...

Mapping the receptor site for α-scorpion toxins on a Na+ channel voltage sensor

The α-scorpions toxins bind to the resting state of Na+ channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ∼73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na+ channels and reveals its mode of interaction with a gating modifier toxin.

The Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins

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 Ile73 and Ile74 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.

BACULOVIRUS-MEDIATED EXPRESSION OF A SCORPION DEPRESSANT TOXIN IMPROVES THE INSECTICIDAL EFFICACY ACHIEVED WITH EXCITATORY TOXINS

The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti‐insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild‐type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.

Sea anemone toxins affecting voltage-gated sodium channels - molecular and evolutionary features

The venom of sea anemones is rich in low molecular weight proteinaceous neurotoxins that vary greatly in structure, site of action, and phyletic (insect, crustacean or vertebrate) preference. This toxic versatility likely contributes to the ability of these sessile animals to inhabit marine environments co-habited by a variety of mobile predators. Among these toxins, those that show prominent activity at voltage-gated sodium channels and are critical in predation and defense, have been extensively studied for more than three decades. These studies initially focused on the discovery of new toxins, determination of their covalent and folded structures, understanding of their mechanisms of action on different sodium channels, and identification of the primary sites of interaction of the toxins with their channel receptors. The channel binding site for Type I and the structurally unrelated Type III sea anemone toxins was identified as neurotoxin receptor site 3, a site previously shown to be targeted by scorpion alpha-toxins. The bioactive surfaces of toxin representatives from these two sea anemone types have been characterized by mutagenesis. These analyses pointed to heterogeneity of receptor site 3 at various sodium channels. A turning point in evolutionary studies of sea anemone toxins was the recent release of the genome sequence of Nematostella vectensis, which enabled analysis of the genomic organization of the corresponding genes. This analysis demonstrated that Type I toxins in Nematostella and other species are encoded by gene families and suggested that these genes developed by concerted evolution. The current review provides a brief historical description of the discovery and characterization of sea anemone toxins that affect voltage-gated sodium channels and delineates recent advances in the study of their structure-activity relationship and evolution.

Diversification of neurotoxins by C-tail ‘wiggling’: a scorpion recipe for survival

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 ven¬omous animals such as sea anemones, cone snails, scorpions, and snakes show multiple routes for active site diversification, each maintaining a typical con¬served 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 consid¬erably, 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.—Gurevitz, M., Gordon, D., Ben‐Natan, S., Turkov, M., Froy, O. Diversification of neurotoxins by C‐tail ‘wiggling’: a scorpion recipe for survival. FASEB J. 15, 1201–1205 (2001)

Further enhancement of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively

We have studied whether the cooperative insecticidal effect of certain scorpion toxin pairs, namely either a combination of excitatory and depressant, or alpha and depressant scorpion toxins, would improve the efficacy of Autographa californica nucleopolyhedrovirus (AcMNPV) over a virus expressing only a single toxin, towards Heliothis virescens, Helicoverpa armigera, and Spodoptera littoralis larvae. The best result was achieved by combined expression of the excitatory toxin, LqhIT1, and the depressant toxin, LqhIT2, that provided an ET50 value of 46.9 h on H. virescens neonates, an improvement of 40% over the efficacy of wild‐type AcMNPV, and of 18% and 22% over baculoviruses that express each of the toxins independently. These results demonstrate that significant improvement in efficacy of recombinant baculoviruses is obtainable with toxins that exhibit a cooperative effect, and may contribute to employ baculoviruses to replace hazardous chemicals in insect control.

Membrane potential modulators: a thread of scarlet from plants to humans

The preservation along evolution of specific core motifs in proteins of diverse functions and taxonomic origins pinpoints a possible developmental advantage at the structural level. Such a preservation was observed in a group of membrane potential modulators including plant γ‐thionins, scorpion toxins, insect and scorpion defensins, bee venom apamin and MCD peptide, snake sarafotoxins, and human endothelins. These substances are short polypeptides of various lengths and nonhomologous sequences that affect organisms of distant phyla. Despite the structural differences, comparative analysis reveals commonality at three levels: 1) effect on membrane potential; 2) a common cysteinestabilized α‐helical (CSH) motif; and 3) similar gene organization (except for insect defensins), i.e., an intron that splits a codon toward the end of the leader sequence. We thus propose that these modulators, divided into two groups differing in their CSH motif orientation, have either diverged from two independent ancestors or have evolved by gene diversification via exon shuffling and subsequent modifications. To enforce protein synthesis through the secretory pathway and enable disulfide bond formation and secretion, insertion sites downstream of preexisting leader sequences have been a prerequisite. What seems advantageous for evolution, may also be exploited in attempts to ‘accelerate evolution’ by protein design using the conserved CSH core as a suitable scaffold for reshaping molecular exteriors.—Froy, O., Gurevitz, M. Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 (1998)

Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae

The Leiurus quinquestriatus hebraeus alpha anti‐insect toxin (LqhαIT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional LqhαIT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti‐insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti‐insect selectivity.

Positions under positive selection - key for selectivity and potency of scorpion α-toxins

Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.