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Results: 5

1.
Figure 2

Figure 2. The catalytic domain of ExoU displays an α/β hydrolase fold.. From: Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa.

(A) Ser142 is in close proximity to the Gly111–113 elbow, which acts as an oxyanion hole for the phospholipase-catalyzed reaction. The second member of the Ser-Asp catalytic dyad, Asp344, is located in a flexible region that is not traceable in the electron density map. The overall arrangement of the core region of ExoU's catalytic domain is reminiscent of that of human cPLA2 (B), which also harbors the catalytic aspartate in a moveable ‘cap’ (in violet).

Claire Gendrin, et al. PLoS Pathog. 2012 April;8(4):e1002637.
2.
Figure 4

Figure 4. Direct visualization of Ub-ExoU by UbFC.. From: Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa.

(A) Schematic representation of the plasmid constructs used for UbFC. The genes from which the different fragments were amplified are indicated within the boxes, and the number of residues of each fragment is indicated in parentheses. (B) ExoU-S142A-VC and VN-Ub (upper panel) or ExoU-S142A-VC and VN-Ub Mut (lower panels) were co-expressed in HeLa cells, and fluorescence complementation was observed 36 h after transfection. Immunodetection of fusion proteins with the anti-HA and the anti-FLAG antibodies was performed to confirm protein expression Bars, 12 µm. Pearson's correlation coefficient for Fig. 4B top=0.727.

Claire Gendrin, et al. PLoS Pathog. 2012 April;8(4):e1002637.
3.
Figure 5

Figure 5. Ub-ExoU is targeted to endosomes.. From: Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa.

(A) Co-localization of Ub-ExoU with endosomal markers. ExoU S142A-VC was co-transfected with VN-Ub (upper panel) or with VN-Ub K63 (bottom panel). 36 h later, cells were stained with the anti-EEA1, or treated with lysotracker red. Pearson's correlation coefficients correspond to 0.692, 0.570, and 0.801 the top left, top right, and bottom panels, respectively. (B). HeLa cells were infected for 3 h with PaΔST+ExoU S142A (upper panel), PaΔST+ExoU S142A-K178R or PaΔST+ExoU S142A Δ679–683 (bottom panel), fixed and immunolabeled with anti-ExoU and anti-EEA1 antibodies. Single two-channel confocal images are presented. The Pearson's correlation coefficient for EEA1-ExoU=0.213. Bars, 12 µm.

Claire Gendrin, et al. PLoS Pathog. 2012 April;8(4):e1002637.
4.
Figure 1

Figure 1. The crystal structure of ExoU in complex with its chaperone SpcU.. From: Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa.

(A) Schematic diagram of the ExoU construct used in this work and limitations of different domains. The first and last ExoU residues observed in the electron density map are Gly53 and Glu685. The catalytic domain is inserted within the first and second bridging subdomains. (B) ExoU folds into three distinct domains and its N-terminal β-strand is paired with SpcU's β-sheet. The bridging domain (yellow) contains elements that are both N-terminal and C-terminal to the catalytic domain (blue). The membrane-binding domain makes few contacts with the other domains of ExoU. (C) SpcU (green) is a type IA T3SS chaperone. The N-terminus of ExoU, packs as a β-strand against the central 5-stranded SpcU sheet. For clarity, only the asymmetric unit is shown.

Claire Gendrin, et al. PLoS Pathog. 2012 April;8(4):e1002637.
5.
Figure 3

Figure 3. Membrane-binding, ubiquitination, and catalysis are structurally intertwined.. From: Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa.

(A) Mutations in the C-terminus of ExoU [31], [32], [37]–[40] that abrogate membrane binding and cytotoxicity are all located within the four-helical bundle (red). This region is located on the same face of the molecule as the catalytic region, as well as Lys178 (left). (B) Residues 679–683 are exposed to solvent, in a potential appropriate orientation for bilayer recognition. (C) Protein-lipid overlays of wt ExoU, ExoU-S142A and ExoU-Δ679–683 reveal that the C-terminal region is essential for a strong, specific interaction with PI(4,5)P2. TG, triglyceride; DAG, diacylglycerol; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; CL, cardiolipin; PI, phosphatidyl-inositol; PI(4)P, phosphatidylinositol 4 phosphate; PI(4,5)P2, phosphatidylinositol (4,5) di-phosphate; PI(3,4,5)P3, phosphatidylinositol (3,4,5) triphosphate; chol, cholesterol; SM, sphingomyelin; SGC, 3 sulfogalactosylceramide. Blank=no lipid spotted.

Claire Gendrin, et al. PLoS Pathog. 2012 April;8(4):e1002637.

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