title: Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function.
abstract: Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication.
authors: Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0366, USA.
authors: Audrey
authors: A
authors: Fischer
authors: Mauricio
authors: M
authors: Montal
mesh_terms: D000818:Animals; D054045:Biological Warfare Agents; D001905:Botulinum Toxins / Q000737:chemistry*; D002460:Cell Line; D003014:Clostridium botulinum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D009498:Neurotoxins / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D054045:Biological Warfare Agents; D018832:Molecular Chaperones; D009498:Neurotoxins; D001905:Botulinum Toxins
keywords: Antibotulinal agents; BTD; Biological weapons; BoNT; Botulism; HC; LC; LC-TD; Modular nanomachine; P(o); Protein translocase; RBD; TCEP; TD; V(½); beltless translocation domain; beta-mercaptoethanol; botulinum neurotoxin; channel open probability; heavy chain; light chain; light chain-translocation domain; pH gradient across the cis and trans compartments; receptor binding domain; single channel conductance; t(1/2); the half time for completion of translocation; the voltage at which P(o) = 0.5; translocation domain; tris-(2-carboxyethyl) phosphine; ΔpH; βME; γ
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