ABSTRACT
Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.
Subject(s)
Bacterial Toxins/chemistry , Clostridium perfringens/pathogenicity , Gas Gangrene , Metalloproteins/chemistry , Type C Phospholipases/chemistry , Zinc , Amino Acid Sequence , Bacterial Toxins/metabolism , Binding Sites , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Computer Simulation , Crystallography , Hemolysin Proteins/chemistry , Humans , Male , Membranes/metabolism , Metalloproteins/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Second Messenger Systems , Sequence Homology, Amino Acid , Synchrotrons , Type C Phospholipases/metabolismABSTRACT
The alpha-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man. The gene encoding the alpha-toxin has been cloned into E. coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity. The two strains of alpha-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant. The alpha-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K. CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to dmin = 1.90 A. The characteristics of the NCTC8237 form I crystals have already been reported. The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 A, alpha = beta = gamma = 90 degrees, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to maximum resolutions of 1.85 and 2.1 A for the CER89L43 and the NCTC8237 strains, respectively.
