doi:10.1016/j.jmb.2006.09.050 
Copyright © 2006 Elsevier Ltd All rights reserved.
The HC Fragment of Tetanus Toxin forms Stable, Concentration-dependent Dimers via an Intermolecular Disulphide Bond
Omar Qazi1, Barbara Bolgiano2, Dennis Crane2, Dmitri I. Svergun3, 4, Petr V. Konarev3, 4, Zhong-Ping Yao5, Carol V. Robinson5, Katherine A. Brown1 and Neil Fairweather1, 
, 
1Division of Cell and Molecular Biology, Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK
2Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
3European Molecular Biology Laboratory, Hamburg Outstation, Notkestraβe 85, D-22603 Hamburg, Germany
4Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
5Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK
Received 30 June 2006;
revised 18 September 2006;
accepted 19 September 2006.
Edited by M. Moody.
Available online 23 September 2006.
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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 (HC) 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 HC undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect HC oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of HC 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 HC in solution. We propose a possible mechanism for HC oligomerisation and discuss how this may relate to toxicity.
Keywords: tetanus toxin; oligomerisation; ganglioside binding
Abbreviations: TeNT, tetanus neurotoxin; CNT, clostridial neurotoxin; SEC, size exclusion chromatography; MALLS, multi-angle laser light scattering; ESI-ToF, electrospray ionisation time-of-flight; SAXS, small angle X-ray scattering
Figure 1. Location of cysteine residues in the TeNT HC protein. (a) Line drawing of TeNT HC showing positions of cysteine residues. (b) Ribbon structure of TeNT HC, showing the two protein domains: HCC and HCN.11 The four cysteine residues are shown in green. Cys869 and Cys1093 form an intramolecular disulphide bond in the N-terminal domain of the protein. The structure was displayed using Swiss PDB viewer and the image rendered using POV-Ray.
Figure 2. SDS–PAGE of HC proteins separated under both reducing and non-reducing conditions. Under reducing conditions, all proteins run as a single band at approximately 50 kDa. Under non-reducing conditions, the appearance of multiple forms in all proteins is evident, with the exception of HCCys869Ala. M, molecular mass markers.
Figure 4. Molar mass versus volume plot of Zimm-determined weight-average molecular mass values from SEC/MALLS of HCWT protein. The protein was eluted from a TSK 5000PWXL column in PBS (pH 7.4). The continuous line is the refractive index trace; the individual points are molar mass values determined at each data point.
Figure 5. Analysis of HC proteins by 10–15% native PAGE in the presence of increasing concentrations of DTT (1, 5 and 10 mM). HCWT, polydispersed protein; HCWT-D, dimeric protein purified by SEC. The open and filled arrowheads indicate the migration position of dimeric and monomeric HC, respectively.
Figure 6. ESI-ToF mass spectra of (a) polydispersed HCWT protein; (b) monomeric HCWT protein; (c) and (d) dimeric HCWT protein. Monomeric and dimeric forms of the protein were obtained by SEC and frozen. Upon thawing samples were concentrated and subjected to electrospray ionisation (cone voltage 150 V). The dimeric protein in (c) was stored on ice for 24 h prior to analysis, whereas the sample in (d) was analysed immediately after concentration.
Figure 7. SAXS analysis of HCWT. For monomeric HC (curve 1), the computed fit from the crystallographic model is displayed as open triangles, the fit from a mixture of monomers and dimers shown as a continuous line. For dimeric HC (curve 2), the continuous line is the fit from the ab initio model and open triangles represent the fit from the dimeric model obtained by rigid body refinement. Curves 3–6 are polydispersed HC at solute concentrations of 2.5, 5.0, 10.0 and 18.0 mg ml− 1, respectively, and the continuous lines represent fits from the mixture of monomers and dimers obtained by the program OLIGOMER. Successive curves are displaced down by one logarithmic unit for clarity. Dots, experimental scattering data; continuous lines and symbols, fits from the models. The plot displays the logarithm of the scattering intensity as a function of momentum transfer.
Figure 8. Ab initio bead model of dimeric HCWT obtained by DAMMIN (grey semitransparent spheres) superimposed with the model constructed by SASREF using automated rigid body modelling. Monomers are displayed as blue and red Cα lines; the residue Cys869 in each monomer is indicated by a green sphere. The top left view is along the 2-fold axis; the right and bottom orientations are rotated counter-clockwise around the vertical and horizontal axis, respectively. In this model, interactions occur between the N-terminal domains of two monomers.
Table 1.
GT1b ganglioside binding activities of proteins
Polydispersed HCWT and mutant proteins were assayed for ganglioside binding by ELISA.
Table 2.
Composition of oligomeric HCWT protein calculated by SEC
a Calculated from the SEC UV trace: mAu x ml.
b Calculated from fraction concentrations using the BCA assay.
Table 3.
Overall parameters of HCWT protein samples calculated from SAXS data
Rg is the radius of gyration, Dmax is maximum size of particles, VPorod is excluded volume, MMexp is the molecular mass of the solute, vm and vdim = 1-vm are volume fractions of monomers and dimers, respectively, computed by the program OLIGOMER (see the text). For polydisperse HC samples, discrepancy χ values from OLIGOMER are presented; for monomer HC sample χ provided by the crystallographic model and of the best OLIGOMER fit are given; for dimeric HC, χ values of the fit from the ab initio model and the fit from rigid body refinement are shown.
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100 amino acid residue segments were isolated with the ability to assemble into active homo-pentamers. The topology of most of these segments follows a “hidden” module that differs from the modules observed in wild-type Tachylectin-2, yet their biophysical properties and sugar binding activities resemble the wild-type's. Since the pentamer's molecular mass is twofold higher than the wild-type (
