CRYSTALLOGRAPHIC STUDIES OF ALPHA-TOXIN (PHOSPHOLIPASE C) FROM CLOSTRIDIUM PERFRINGENS. A. K. Basak1, J. T. Eaton1, D. S. Moss1, R. W. Titball2, 1Department Of Crystallography, Birkbeck College, UK, 2Chemical & Bological Defence Establishment, Porton Down, UK.
A wide variety of gram-positive and gram-negative bacteria produce phospholipase C s and these enzymes have markedly different biophysical properties. The enzymes hydrolyse different phospholipids with varying efficiencies and only some of them have haemolytic and lethal properties. The enzyme alpha-toxin of Clostridium perfringens is the most toxic phospholipase C characterized to date, but in spite of that it is still not clear why this enzyme (and some other phospholipases C) is toxic whereas others such as phosphatidylcholine preferring phospholipase C (PC-PLC) from Bacillus cereus, are non-toxic. The N-terminal two-thirds of the protein (1-249 residues) show amino acid sequence homology with the entire B. cereus PC-PLC. It is also known that the C-terminal domain of the protein confers the haemolytic and lethal properties of the phospholipase C.
In order to investigate the molecular basis of the toxicity of the alpha-toxin we are currently determining the crystal structure of the enzyme. The protein is composed of a single polypeptide chain of 370 amino acid residues and has a molecular weight of 42.5kDa The protein has been expressed in E. Coli and purified in two different strains. Three different crystal forms suitable for X-ray diffraction analysis have been grown from these strains.
Initial attempts to solve the structure by molecular replacement methods using the known B. cereus phospholipase C structure as a model were not successful. Subsequently phases have been determined (from one of these three crystal forms) using three different heavy atom derivatives and an initial solvent-flattened electron density map at 3.5Å resolution shows the secondary structural elements of the molecule. Interpretation of the electron density map, phase extension are currently in progress to provide an accurate structure.