A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1

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.