THE CARBOXY-TERMINAL C-2-LIKE DOMAIN OF THE ALPHA-TOXIN FROM CLOSTRIDIUM-PERFRINGENS MEDIATES CALCIUM-DEPENDENT MEMBRANE RECOGNITION

The lethal, cytolytic alpha-toxin (phospholipase C) of Clostridium per fringens consists of two distinct modules: the larger N-terminal domai n catalyses phospholipid hydrolysis, and its activity is potentiated b y a smaller C-terminal domain. Calcium ions are essential for the bind ing of alpha-toxin to lipid films. Sixteen alpha-toxin variants with s ingle amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were consid erably less active than native alpha-toxin under calcium-limiting cond itions. Replacement of Thr-272 by Pro diminished phospholipase C activ ity, severely affected haemolysis and platelet aggregation and perturb ed a surface-exposed conformational epitope. The results of sequence c omparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C-2 beta-barrel domains, wh ich are often involved in membrane interactions, and that the function ally important substitutions are clustered at one extremity of the dom ain. The combined findings suggest that the C-terminal region of alpha -toxin mediates interactions with membrane phospholipids in a calcium- dependent manner. Mutations to this domain may account for the natural lack of toxicity of the alpha-toxin homologue, phospholipase C of Clo stridium bifermentans.