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, 50 (6), 917-9

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-activated Kinase

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Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-activated Kinase

Katherine L Germane et al. Biochemistry.

Abstract

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

Figures

Figure 1
Figure 1
EspG (A.) and VirA (B.) shown as ribbon diagrams with the NT domain colored green, the central sheet orange, and the helical domain salmon. The structures are oriented so that the central sheet and helical domain align and highlights the rotation of the N-terminal domain. Secondary structural elements, and domains are labeled in (B.). Features of EspG described in the text are labeled in (A.).
Figure 2
Figure 2
Surface rendering of EspG, colored by residue conservation as shown. Panel (A.) shows EspG oriented as in figure 1, the residues that align to the VirA cleft are highly variable amongst EspG, EspG2, and VirA family members. (B.) EspG viewed along the arrow in panel (A.). A continuous stripe of highly conserved residues are observed in (A.) and continued into (B.). A deep, highly conserved pocket, the rim of which is formed by residues from α4 and α9, is seen in (B.). Residues D205 and R208 form much of the entrance to the pocket and are strictly conserved.
Figure 3
Figure 3
PAK-PBD pull-down assay. (A.) Cells expressing equivalent amounts of flag-tagged EspG and EspG_DR-AA were incubated with PBD-beads. EspG remained bound to PBD after 3 washes, whereas EspG_DR-AA was predominately washed off. (B.) Neither EspG nor EspG_DR-AA bind to GST-beads. (C.) Direct binding between purified EspG and GST-PBD indicates that no other cellular proteins are involved in mediating the EspG-PBD interaction. (D.) Expression of EspG, and to a lesser extent EspG_DR-AA, results in loss of active (phosphorylated) PAK1.

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