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T

etanus toxin (TeNT) and botulinum toxin (BoNT)

are amongst the most potent toxins known. They

are produced by Clostridial bacteria (C. tetani and C.

botulinum, respectively) and the purpose is to kill the host

via paralysis and suffocation.

The toxins are structurally related (about 50 sequence

identity) and functionally related. However, TeNT targets

the neurons of the spinal cord via trans-synaptic migration,

whereas BoNT targets secondary neurons.

The mechanism of these toxins consists of 4 stages,

cell binding, vesicular internalisation, cytoplasmic

translocation and finally proteolytic cleavage of the

substrate by the L-chain [1]. It is in order to understand

these functions that we have undertaken the highresolution structure of Tetanus toxin HC.

TeNT is synthesised as a single polypeptide chain

which undergoes cleavage to produce a mature toxin

consisting of the N-terminal 50 kD fragment linked via a

disulphide bond to the 100 kD C-terminal fragment (H

chain). The 50 kD fragment of the C-terminal portion of

the H chain (known as HC

or fragment C) is responsible for

ganglioside binding, which is essential for binding of the

toxin to neuronal cells.

Crystals of HC were grown in conditions similar to

those published previously [2] with the modification of the

addition of PEG 4K and 1 MPD. Cryo-cooled data collection

was performed at Daresbury stations 7.2, 9.5 and 9.6:

3.0 UAc data,

3.0 PtCl data

2.5 HgAc + CH3HgCl data

3.0 HgAc + CH 3HgCl (different soak-time and

wavelength)

1.8 data (Daresbury 7.2)

The 2.5 Hg dataset (compared to the 2.3 native)

provided the position of the leading 3 Hg peaks. The other

11 sites were found from difference Fouriers and similar

maps from SHARP. Density modification (DM) provided a

map in which most of the model was traced. A few

remaining loops were traced with the aid of a picture of a

CA-representation provided in [3].

The high resolution native showed considerable lack

of isomorphism compared to the lower (2.3) data (scaling

R-factors typically 20-30). Therefore, AMORE [4] was then

used to reposition the molecule in the unit cell of the high

resolution data. The refinement proceeded conventionally,

using REFMAC [5, 6] and ARP [7] finding 3 glycerol

molecules, 400 water molecules and geometry judgedacceptable (from the output of PROCHECK [8]) for a typical

1.8 structure.

Structural conclusions: Tetanus toxin HC consists of

two domains, the N-terminal domain is a -sandwich and

the C-terminal domain is a -trefoil. The -trefoil contains

the ganglioside binding sites. Several glycerol molecules

have been observed in the crystal structure as has been seen

previously, which may indicate the carbohydrate

binding site.

Tetanus Toxin HC

P. Emsley, N.W. IsaacsDepartment of Chemistry, University of Glasgow, Glasgow, G12 8QQN. FairweatherImperial College, University of LondonI.G. CharlesThe Cruciform Project, Imperial College, University of London

Scientific ReportsPROTEIN CRY S TA L L O G R A P H Y

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Figure 1. Tetanus toxin Hc

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PROTEIN CRY S TA L L O G R A P H Y

References

[1] C. Montecucco, (1986). How do tetanus and botulinum toxins

bind to neuronal membranes? Trends in Biochem. Sci. 11,

pp314-317.

[2] M. Anderson, I. G. Charles, P. Emsley, N. Fairweather, G.

McDermott, N. W. Isaacs, (1993). J. Mol. Biol. 230, pp673-674.

[3] T. C. Umland, L. M. Wingert, S. Swaminathan, W. Furey, J. J.

Schmidt, M. Sax, (1997). Structure of the receptor binding

fragment H of tetanus neurotoxin. Nature Structural Biology 4,

pp788-792.

[4] J. Navaza, (1994). Amore: an automated package for molecular

replacement. Acta Crystallographica A, pp157-163.

[5] G. N. Murshudov, A. A. Vagin, E. J. Dodson, (1997). Refinement

of macromolecular structures by the maximum-likelihood method.

Acta Crystallographica D, pp240-255.

[6] Collaborative Computational Project, Number 4, (1994). The ccp4suite: Programs for protein crystallography. Acta.

Crystallographica D, pp760-763.

[7] V. S. Lamzin K. S. Wilson, (1993). Automated refinement of

protein models. Acta Crystallographica D, pp129-147.

[8] R. A. Laskowski, M. W. MacArthur, D. S. Moss, J. M. Thornton,

(1993). A program to check the stereochemical quality of protein

structures. J. Appl. Crystallogr. 26, pp283-291.