We also performed the following mutations for the amino acid residues surrounding the tryptophans. Because some of the amino acids adjacent to the three tryptophan residues carry electrical charges, we changed the charge in each amino acid residue. We changed two residues, E306 and D308, from acidic to basic amino

acids by replacement with arginine (E306R and D308R). We replaced the residue K310 with glutamic acid in order to change from basic to acidic type (K310E). We also substituted the residue V312 with alanine to maintain hydrophobicity and no electric charge (V312A). We constructed mutant toxins in which we replaced residue N302, the most amino-terminal domain side in the tryptophan-rich region, with alanine (N302A). Wild-type and mutant alpha-toxins were expressed

in E. coli BL21 and purified by affinity chromatography. SDS–PAGE detected every purified mutant toxin at the expected positions find more and each of their secondary structures was similar to that of wild-type toxin according to far-ultraviolet (190–260 nm) circular dichroism LY2606368 mouse spectral analysis (data not shown). As shown in Table 3, the cytotoxic activities (EC50) of mutant toxins were compared with that of wild-type toxin. We found that the EC50 of W307F/W309F/W311F and W307A/W309A/W311A were >640 ng/mL, indicating that the cytotoxic activity of alpha-toxin decreased remarkably to below the limit of detection. The

mutants of W307A, W309A and W311A also had marked reduction of cytotoxic activity. Although replacements of W307 and W311 with phenylalanine decreased the cytotoxic activities (207 and 113 ng/mL), they did not completely abolish them. Interestingly, replacement of W309 with phenylalanine did not greatly reduce cytotoxic activity. The mutant of W309F retained the same activity as the wild type. In the case of amino acid substitutions surrounding the three tryptophan residues, only D308R caused a decrease in cytotoxic Elongation factor 2 kinase ability (127 ng/mL). The cytotoxic activities of E306R, K310E, K310R, V312A and N302A did not change in comparison with that of the wild type. To determine whether the tryptophan-rich region plays an important role in the binding of alpha-toxin to cell membranes, we used a toxin overlay assay to examine the binding activities of mutant toxins to detergent-insoluble proteins from Vero cells. After lysis with 1% Triton X-114, we separated Vero cells into detergent-soluble and -insoluble fractions by centrifugation. As shown in Figure 2a, we observed a specific band with a molecular mass of about 34 kDa in the detergent-insoluble fraction using a toxin overlay assay with wild-type alpha-toxin. In previous studies, we reported that alpha-toxin selectively binds to GPI-anchored proteins detected in the detergent-insoluble fractions from various cell lines [12, 25].