title: Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?
abstract: Botulinum neurotoxin, produced by Clostridium botulinum as a approximately 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The approximately 50- and approximately 100-kDa chains of the dichain molecule remain held together by an interchain disulfide bridge and noncovalent interactions. The neurotoxin binds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the approximately 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state. The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing. The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and DasGupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-830), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel.
authors.affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee, 37235, USA.
authors.forename: P F
authors.initials: PF
authors.lastname: Flicker
authors.forename: J P
authors.initials: JP
authors.lastname: Robinson
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism; D019274:Botulinum Toxins, Type A; D003014:Clostridium botulinum / Q000737:chemistry; D020285:Cryoelectron Microscopy; D003460:Crystallization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000378:metabolism; D005732:Gangliosides; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure; D008051:Lipid Bilayers; D008666:Metalloendopeptidases / Q000737:chemistry; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D004220:Disulfides; D005732:Gangliosides; D007473:Ion Channels; D008051:Lipid Bilayers; C096323:rimabotulinumtoxinB; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D004229:Dithiothreitol