Miniaturization of scorpion β-toxins uncovers a putative ancestral surface of interaction with voltage-gated sodium channels

The bioactive surface of scorpion beta-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved beta alpha beta beta 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 beta-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 beta-toxins (Delta Delta Bj-xtrIT and Delta Delta Css4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, Delta Delta Bj-xtrIT and Delta Delta Css4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion alpha-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion alpha-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of Lqh alpha IT at insect sodium channels. Co-application of Delta Delta Bj-xtrIT or Delta Delta Css4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at Delta Delta Bj-xtrIT and Delta Delta Css4 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 beta-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.