UC Irvine

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (H_N), but the molecular mechanism underlying membrane insertion of H_N remains poorly understood. Here, we report two crystal structures of BoNT/A1 H_N that reveal a novel molecular switch (termed BoNT-switch) in H_N, where buried α-helices transform into surface-exposed hydrophobic β-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of H_N with anionic liposomes, blocked channel formation by H_N, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of H_N that subsequently leads to LC delivery.