Chemical Engineering and Structural and Pharmacological Characterization of the α‑Scorpion Toxin OD1

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure–function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Na_v1.7, as well as Na_v1.4 and Na_v1.6. In addition, OD1 also produced potent β-toxin activity at Na_v1.4 and Na_v1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Na_v1.4 and Na_v1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Na_v1.7 over Na_v1.6, while OD1 K11V was 5-fold more selective for Na_v1.6 than Na_v1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Na_v subtypes.