Positive selection sites (PSSs), a class of amino acid sites with an excess of non-synonymous 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 α-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_vchannels. In this study, we undertook a systematical molecular dissection of nearly all the PSSs newly characterized in the Mesobuthus α-toxin family and a two-residue insertion (¹⁹AlaPhe²⁰) located within a positively selected loop via mutational analysis of α-like MeuNaTxα-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 (Ile⁴⁰ and Leu⁴¹) 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 MeuNaTxα-5 with partially impaired or enhanced insecticide activity, suggesting the potential of PSSs in designing promising candidate insecticides from scorpion α-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.

http://mbe.oxfordjournals.org/content/early/2016/04/04/molbev.msw065