title: Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B.
abstract: Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. BoNTs consist of three essential domains for toxicity: the cell binding domain (Hc), the translocation domain (Hn) and the catalytic domain (LC). A functional derivative (LHn) of the parent neurotoxin B composed of Hn and LC domains was recombinantly produced and characterised. LHn/B crystallographic structure at 2.8Å resolution is reported. The catalytic activity of LHn/B towards recombinant human VAMP was analysed by substrate cleavage assay and showed a higher specificity for VAMP-1, -2 compared to VAMP-3. LHn/B also showed measurable activity in living spinal cord neurons. Despite lacking the Hc (cell-targeting) domain, LHn/B retained the capacity to internalize and cleave intracellular VAMP-1 and -2 when added to the cells at high concentration. These activities of the LHn/B fragment demonstrate the utility of engineered botulinum neurotoxin fragments as analytical tools to study the mechanisms of action of BoNT neurotoxins and of SNARE proteins.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Masuyer
authors.forename: Matthew
authors.initials: M
authors.lastname: Beard
authors.forename: Verity A
authors.initials: VA
authors.lastname: Cadd
authors.forename: John A
authors.initials: JA
authors.lastname: Chaddock
authors.forename: K Ravi
authors.initials: KR
authors.lastname: Acharya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D050987:Vesicle-Associated Membrane Protein 1 / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: D050987:Vesicle-Associated Membrane Protein 1; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A