CONTRIBUTION OF INDIVIDUAL TRYPTOPHAN RESIDUES TO THE STRUCTURE AND ACTIVITY OF THETA-TOXIN (PERFRINGOLYSIN-O), A CHOLESTEROL-BINDING CYTOLYSIN

theta-Toxin (perfringolysin O), secreted by Clostridium perfringens, s hares with other known thiol-activated toxins a conserved undecapeptid e, ECTGLAWEWWR, located in the C-terminal region of the protein and co ntaining the unique cysteine of the molecule. Single and double amino acid substitutions were created in the theta-toxin molecule to investi gate the role of individual tryptophan residues in the lytic activity of theta-toxin. Wild-type and mutant theta-toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified. The relative hemolytic activities of four mutant toxins, eac h with a Trp to Phe substitution outside the common Cys-containing reg ion, were more than 60% that of wild-type theta-toxin. In contrast, mu tant toxins with Phe replacements within the Cys-containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte-membrane-binding activities. The largest reduction in bind ing affinity, more than 100-fold, was observed fur Trp438 mutant toxin s. However, the mutants retain binding specificity for cholesterol and the ability to form arc-shaped and ring-shaped structures on membrane s. These results indicate that the low hemolytic activities of these m utant toxins can be ascribed, at least in part, to reduced binding act ivities. With respect to protease susceptibility and far-ultraviolet c ircular-dichroism spectra, only the W436-->F mutant toxin, showed any considerable difference from wild-type toxin in secondary or higher-or der structures, indicating that Trp436 is essential for maintenance of toxin structure.