title: The carboxy-terminal C2-like domain of the alpha-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition.
abstract: The lethal, cytolytic alpha-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain. Calcium ions are essential for the binding of alpha-toxin to lipid films. Sixteen alpha-toxin variants with single 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 considerably less active than native alpha-toxin under calcium-limiting conditions. Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C2 beta-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. 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 Clostridium bifermentans.
authors.affiliation: Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Guillouard
authors.forename: P M
authors.initials: PM
authors.lastname: Alzari
authors.forename: B
authors.initials: B
authors.lastname: Saliou
authors.forename: S T
authors.initials: ST
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D003470:Culture Media; D013912:Threonine; D014443:Tyrosine; D011392:Proline; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D002118:Calcium