title: 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 approximately 50 kDa light chain (LC) protease is translocated into the cytosol by the approximately 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.
authors.affiliation: Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Audrey
authors.initials: A
authors.lastname: Fischer
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Mushrush
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
authors.forename: Mauricio
authors.initials: M
authors.lastname: Montal
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D016503:Drug Delivery Systems / Q000379:methods; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018832:Molecular Chaperones / Q000378:metabolism / Q000502:physiology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053586:Virus Internalization
chemical_list: D007473:Ion Channels; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A