Botulinum neurotoxin devoid of receptor binding domain translocates active protease.
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.
Fischer
Audrey
A
Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America.
Mushrush
Darren J
DJ
Lacy
D Borden
DB
Montal
Mauricio
M
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
D007473:Ion Channels; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A