THE KEY DTERMINANT IN GAS-GANGRENE: A NOVEL MEMBER OF THE C2 DOMAIN FOLD FAMILY?

C. E. Naylor¹, J. T. Eaton¹, A. Howells², N. Justin¹, D. Moss¹, R. Titball² and A. K. Basak¹

¹Dept of Crystallography, Birkbeck College, Malet Street, England, WC1E 7HX
²CBD, Porton Down, England.

C. perfringens a-toxin is the key virulence determinant in gas-gangrene disease, and has also been implicated in a number of other diseases of man and animals. The toxin is a 370 amino acid zinc metalloenzyme of 43 kDa molecular weight, which has phospholipase C, sphingomylinase and haemolytic activities. In the presence of calcium ions, a-toxin can cleave membrane packed phospholipid. Here, we present the structure of the C. perfringens a-toxin, strain CER89L43. It is a 2 domain structure. The N-terminal, catalyti,c domain (residues 1-246) shows an anticipated structural homology to the non-toxic B. cereus phospholipase C. Both proteins contain 3 bound zinc ions: the residues that ligate the metal ions are completely conserved between the two proteins. There are a number of structural differences between a-toxin and the B. cereus enzyme, however, and these will be described in the talk. The C-terminal domain (residues 255-370) is essential for membrane binding, it shows a strong, unexpected structural analogy to the eukaryotic calcium binding C2 domain fold. We believe this is the first example of such a domain in prokaryotes. The C2 domain is normally found acting as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins, and, interestingly these pathways are perturbed in cells treated with a-toxin. This homology has been useful in defining a possible model for membrane binding, which, when used in comparison with other, structurally analogous, but non-toxic, phospholipases, has illuminated the structural features important in a-toxin for toxicity.

Secondary Structure Cartoon of a-toxin. Zinc ions are shown as black spheres, possible calcium binding sites are highlighted as grey spheres.