FIGURE 1.

ExoU MLD requires specific residues to bind to PI(4,5)P₂. A, recombinant ExoU MLD (residues 503–687) was tested for co-sedimentation in liposome binding assays with various lipids. Liposomes consisting of 30% PC, 20% PE, and 20% cholesterol were supplemented with 30% of the indicated lipids, except for PI(4)P, PI(5)P, PI(4,5)P₂, and PI(3,4,5)P₃, for which 5% was added (the remaining 25% was PC). Each assay was performed at least in triplicate; values are means, and error bars represent S.E. *, p < 0.05 compared with all other groups. The difference between PI(4)P and PI(4,5)P₂ is not significant. PI, phosphatidylinositol. B, recombinant MLD proteins containing single amino acid substitutions (I609N, Q623R, N627I, I654N, R661L, and A678D) or a 5-amino acid insertion (LS608) were tested for binding to PI(4,5)P₂-containing liposomes in co-sedimentation assays. Each assay was performed at least in triplicate; values are means, and error bars represent S.E. WT indicates the wild-type MLD of ExoU, using the PI(4,5)P₂ binding data depicted in A. *, p < 0.001 compared with each variant. No difference between the variants is statistically significant. C, recombinant wild-type (WT) and R661L MLD protein were analyzed for binding to PI(4)P-containing liposomes. *, p < 0.05 relative to R661L variant.