Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

Abstract

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication. Botulinum neurotoxin, widely acknowledged to be the most potent toxin known, is a modular nanomachine and a marvel of protein design. This neurotoxin exploits a modular design to achieve its potent toxicity, which relies on one of its modules—the heavy chain channel—to operate as a specific protein translocating transmembrane chaperone for another of its component modules—the light chain protease. Our study shows that a di-modular protein consisting only of protease and translocation domains is sufficient to translocate active cargo into the cytosol of target cells. The receptor binding domain is dispensable for channel activity or LC translocation; however, it regulates the pH threshold of channel insertion into the membrane. The botulinum neurotoxin modular design embodies a tool for biomolecule delivery to predetermined target cells.