E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

Abstract

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Figure 1

Crystal structure of the OspG/UbcH5c-O∼Ub complex and comparison to PKA.

1. Ribbon representation of the OspG/UbcH5c-O˜Ub crystal structure determined at 2.70 Å. Ub (red) is covalently linked to UbcH5c (green) and associates in an extended conformation with OspG (orange). The OspG active site formed by a cleft between the N-and C-lobes is marked and faces away from the UbcH5c˜Ub conjugate.

2. ‘Open book’ representation of the OspG/UbcH5c-O˜Ub interfaces. Surfaces are colored according to their interacting protein and landmark features are labeled.

3. Ribbon representation of the OspG kinase structure looking into the active site cleft. Secondary structure elements are labeled.

4. Global superposition (Supplementary Fig S1D) of OspG and PKA (PDB ID 1ATP; Zheng et al, 1993) overlay important structural and catalytic residues. OspG residues are labeled and the corresponding PKA residues are shown in Supplementary Fig S1C).

5. Superposition of OspG and PKA highlights differences in the arrangement of helices in the C-terminal lobe of each kinase. OspG helices are labeled. The structure of OspG is shown through the C-terminus whereas PKA has an additional 115 residues that are hidden. All PKA residue numbering used here and in the text matches PDB entry 1ATP.

Figure 2

Global superposition of OspG and PKA overlays the electron density in the OspG active site with PKA ATP and Mn²⁺.

1. 2Fo-Fc composite map (blue) and Fo-Fc difference map (green) plotted at 2σ showing unknown density in the OspG active site.

2. Comparison with structurally aligned PKA (PDB ID 1ATP) suggests the density could be bound ATP/Mn²⁺.

Figure 3

UbcH5c Phe62 is required for complex formation with OspG.

1. UbcH5c F62 (green) binds to a shallow pocket formed by OspG residues Y80, L99, P102, and F154.

2. Input: non-reducing samples of reaction mixtures prior to the addition of GST-OspG showing generation of E2˜Ub conjugates from wild-type UbcH5c, F62A-UbcH5c, and Ube2S. Pulldown: reducing SDS-PAGE shows GST-OspG only forms a high-affinity complex with wild-type UbcH5c˜Ub.

Source data are available online for this figure.

Figure 4

OspG binds free UbcH5c and Ub with weak affinity. A, B ¹H,¹⁵N-TROSY spectra of (A) free UbcH5c and Ub and (B) the UbcH5c-O˜Ub conjugate before and after addition of 1 molar equivalent of GST-OspG (grey and red, respectively). OspG shows weak interactions with free UbcH5c and Ub (A). The perturbed UbcH5c and Ub resonances map to the E3-binding interface of UbcH5c and the region surrounding the I44 hydrophobic surface of Ub as shown in the crystal structure. An enlarged region of the center (inset) shows linewidth broadening of the UbcH5c-O˜Ub spectrum after addition of OspG (B), illustrating a substantial increase in affinity attained via conjugation of UbcH5c and Ub subunits.

Figure 5

Complex formation enhances OspG kinase activity but reduces UbcH5c∼Ub intrinsic reactivity.

1. Raw heat outputs and integrated isotherms for titration of 1 mM ATP-γ-S into 50 μM GST (left), GST-OspG (center), or GST-OspG/UbcH5c-O˜Ub complex (right).

2. (1-5) γ³²P-ATP kinase assay following phosphorylation of GST-OspG and Histone H1. 1) OspG alone, 2) premixed OspG + UbcH5c-O˜Ub, 3) premixed OspG + UbcH5c + Ub (unconjugated), 4) premixed K53M-OspG + UbcH5c-O˜Ub, 5) premixed C127R-OspG + UbcH5c-O˜Ub. The panel below depicts the proteins present in each reaction mixture.

3. OspG stabilizes the UbcH5c˜Ub conjugate. 5 μM samples of purified UbcH5c˜Ub or OspG/UbcH5c˜Ub were reacted with 50 mM Lysine over a 60-min time course. Non-reducing gel samples were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by Western analysis for the N-terminal HA epitope on Ub. Decay of the UbcH5c˜Ub species is shown.

Source data are available online for this figure.

Figure 6

OspG/E2∼Ub binding is important for OspG functions during murine model shigellosis. A–C Mortality (A) (n = 10–25 per group), P = 0.03, gross morphology of cecum and colon (B), and CFU counts (C) in homogenized liver (n = 5–15 per group, P > 0.05) and cecum content (n = 5–15 per group, P > 0.05), at day 3 post oral infection of BALB/c mice with WT, ΔospG, ΔospG+ospG^wt, ΔospG+ospG^mut, and ΔospG+ospG^cd Shigella flexneri (1 × 10⁸ CFU oral gavage). D Representative mouse colon sections showing a low and high magnification (area encompassed by the blue box is shown in 100X panels). The low power image shows the proximal regions on the outside of the roll. The high power images were chosen to show more than one run of crypts. Infected mice show some infrequent patchy mucosal and submucosal infiltration by mononuclear cells though the crypt architecture is mostly left intact. An example where the architecture is lost can be seen in the 100X image of the WT infected animal.