Trimming the Disordered C-terminal Extension of the Protease Inhibitor Hg1 Produces a Potent Kv1.3 Blocker

Animal venom contains a mixture of toxic polypeptides that act in synergy to enable the fast immobilization of prey or deter predators and competitors. This polypeptide arsenal is constantly replenished by recruiting protein scaffolds from various organs into the venom gland. The recruited proteins often retain their ancestral functions but acquire specific modifications associated with novel toxic functions. Here we report an in-depth analysis of Hg1, a Kunitz-fold protease inhibitor with moderate blocking activity on Kv1.3, isolated from the venom gland of the Hadrurus gertschi scorpion. Electrophysiological analyses, supported by MD simulations, suggested that in contrast to canonical K+ blocking peptides, Hg1 does not insert a lysine residue into the channel pore. The crystal structure of the toxin revealed an unusually long, disordered extension at the C-terminal region of the molecule. Deletion of the C-terminal extension increased the potency of the toxin by order of magnitude and restored the voltage sensitivity of the block, a hallmark of the canonical block mechanism. In silico analysis supported the notion that removal of the disordered C-terminal extension has lifted a geometric restraint that prevents the wild-type toxin from effectively blocking Kv1.3. We propose that Hg1 is an evolutionary intermediate between a potent protease inhibitor and a potent K+ channel blocker.