FIGURE 3.

Several residues necessary for the binding of ExoU to PI(4,5)P₂ are conserved in other proteins containing ExoU MLD-like domains. A, alignments of proteins containing ExoU MLD-like domains. The highlighted sequences identify highly conserved residues. Those residues highlighted in gray and blue were critical for ExoU localization in mammalian cells (30) and for binding to PI(4,5)P₂-supplemented liposomes (Fig. 1B). Numbering system refers to ExoU of P. aeruginosa. Accession numbers for the indicated proteins in order are as follows: O34208, YP_003039880.1, YP_006326405.1, ZP_07262921.1, ZP_10142741.1, ZP_10438440.1, ZP_11185695.1, ZP_10426928.1, and ZP_18329767.1. B, recombinant putative MLD proteins from ExoU_{P. flu} and ExoU_{P. asy} (residues 453–639 and 495–676, respectively) were purified and assayed for liposome binding. Liposomes consisted of the same composition as depicted in Fig. 1, except that 5% PI/25% PC-supplemented liposomes were also tested. C, arginine-to-leucine substitutions corresponding to R661L in ExoU_{P. aer} were generated in ExoU_{P. flu} and ExoU_{P. asy} recombinant MLD proteins. These proteins were purified and were tested for binding to liposomes supplemented with 5% PI(4,5)P₂. The results for “WT,” which refers to wild-type protein, are the same as depicted in B. Each assay was performed in triplicate; values are means, and error bars represent S.E. *, p < 0.001 for PI(4,5)P₂ compared with other lipids and wild-type compared with arginine-to-leucine substitution proteins.