Abstract

Many spider venom-peptides are known to modulate the activity of the voltage-gated sodium channel NaV1.7, which has emerged as a promising analgesic target. A class of spider venom-peptides (NaSpTx1) in particular have been found to potently inhibit NaV1.7 (nanomolar IC50), and shown to produce analgesic effects in animals. However, one member of this family, μ TRTX Hhn2b (Hhn2b), does not inhibit mammalian NaV channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b leading us to identify two non-pharmacophore residues that contribute to hNaV1.7 inhibition by non-overlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNaV1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom-peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.