title: The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond.
abstract: Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.
authors.affiliation: Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK.
authors.forename: Omar
authors.initials: O
authors.lastname: Qazi
authors.forename: Barbara
authors.initials: B
authors.lastname: Bolgiano
authors.forename: Dennis
authors.initials: D
authors.lastname: Crane
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Petr V
authors.initials: PV
authors.lastname: Konarev
authors.forename: Zhong-Ping
authors.initials: ZP
authors.lastname: Yao
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Brown
authors.forename: Neil
authors.initials: N
authors.lastname: Fairweather
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005732:Gangliosides; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013744:Tetanus Toxin / Q000737:chemistry* / Q000378:metabolism*; D014965:X-Rays
chemical_list: D005732:Gangliosides; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D003545:Cysteine