Target-Driven Positive Selection at Hot Spots of Scorpion Toxins Uncovers Their Potential in Design of Insecticides

Positive selection sites (PSSs), a class of amino acid sites with an excess of nonsynonymous to synonymous substitutions, are indicators of adaptive molecular evolution and have been detected in many protein families involved in a diversity of biological processes by statistical approaches. However, few studies are conducted to evaluate their functional significance and the driving force behind the evolution (i.e., agent of selection). Scorpion alpha-toxins are a class of multigene family of peptide neurotoxins affecting voltage-gated Na+(Na-v) channels, whose members exhibit differential potency and preference for insect and mammalian Na-v channels. In this study, we undertook a systematical molecular dissection of nearly all the PSSs newly characterized in the Mesobuthus alpha-toxin family and a two-residue insertion ((19)AlaPhe(20)) located within a positively selected loop via mutational analysis of alpha-like MeuNaTx alpha-5, one member affecting both insect and mammalian Na-v channels. This allows to identify hot-spot residues on its functional face involved in interaction with the receptor site of Na-v channels, which comprises two PSSs (Ile(40) and Leu(41)) and the small insertion, both located on two spatially separated functional loops. Mutations at these hot-spots resulted in a remarkably decreased anti-mammalian activity in MeuNaTx alpha-5 with partially impaired or enhanced insecticide activity, suggesting the potential of PSSs in designing promising candidate insecticides from scorpion alpha-like toxins. Based on an experiment-guided toxin-channel complex model and high evolutionary variability in the receptor site of predators and prey of scorpions, we provide new evidence for target-driven adaptive evolution of scorpion toxins to deal with their targets' diversity.