Expression and Mutagenesis of the Sea Anemone Toxin Av2 Reveals Key Amino Acid Residues Important for Activity on Voltage-Gated Sodium Channels

Abstract

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na v s) 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 Na v 1.5 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 alphatoxins 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 Na v s varies.

Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels

“…In a very recent paper by Moran et al (Moran et al, 2006), the authors try to resolve the bioactive surface of ATX-II. To this end, they established an efficient expression system for this toxin and mutagenized it throughout.…”

Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

“…Residues V2, L5, D9, N16, L18 and I41 are pivotal amino acids for toxicity to blowfly larvae and for binding to cockroach neuronal membranes. The information from these mutants may be applicable to other insect orders (Moran et al, 2006).…”

Evolution and diversification of the cnidarian venom system

“…To understand the surface accessibility of positively selected and episodically diversifying sites, we computed the accessible surface area (ASA) ratio for all sites in the homology models of these neurotoxins. Our analyses revealed that all four episodically diversifying sites, with an ASA ratio in the range of 60-100 (ASA ≥ 50 is characteristic of surface-exposed sites) in the secreted region of NaTxs, were surface exposed (Supplementary Table S2 Because neurotoxins from N. vectensis (Nv1) and H. carlgreni (Halcurin) are characterised by domains similar to both type I and type II NaTxs, they have been previously suspected to be among the most basal sea anemone neurotoxins [172,173]. Because phylogenetic analyses in this study resolved type I and type II NaTxs into distinct clades, we computed omega values for each of these clades independently.…”

“…Thus, positive selection has strongly shifted the pharmacological properties of the toxin between targets but does not result in the complete loss of activity, which could have a deleterious effect on the fitness of the animal. We also identified a few functionally important sites in other classes of neurotoxins and PFTs that experienced episodic adaptation (Figures 2-2-2-5): 1) Site 2 in NaTxs which was shown to be responsible for a moderate decrease in the binding affinity to insect Nav channels upon mutagenesis (V2A) [173]; 2) sites 54, 117, and 144 in actinoporins that are responsible for binding to cell membranes; and 3) the site in hydralysins shown to result in slight reduction (2.5-fold) of paralytic and hemolytic activity upon mutation (G129E) [58]. Additionally, sites that were shown to have minor effects on toxicity were also found to be positively selected (e.g., sites 12 and 40 in NaTx; Figure 2-1; [173].…”

“…We also identified a few functionally important sites in other classes of neurotoxins and PFTs that experienced episodic adaptation (Figures 2-2-2-5): 1) Site 2 in NaTxs which was shown to be responsible for a moderate decrease in the binding affinity to insect Nav channels upon mutagenesis (V2A) [173]; 2) sites 54, 117, and 144 in actinoporins that are responsible for binding to cell membranes; and 3) the site in hydralysins shown to result in slight reduction (2.5-fold) of paralytic and hemolytic activity upon mutation (G129E) [58]. Additionally, sites that were shown to have minor effects on toxicity were also found to be positively selected (e.g., sites 12 and 40 in NaTx; Figure 2-1; [173]. These findings suggest that sites that are relieved of purifying selection pressures and those responsible for toxicity constitute the evolutionary hotspots in cnidarian toxins, as long as mutations do not result in the complete loss of toxicity.…”