Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Abstract

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUB_GEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUB_GEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUB_GEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

Keywords: Orientia tsutsugamushi; Rac1; X-ray crystallography; guanine nucleotide exchange factor; scrub typhus.

Fig. 1.

OtDUB_275–1369 is toxic in yeast and binds multiple proteins in HeLa cell lysates. (A) Cartoon diagram of various OtDUB fragments used for ectopic expression in yeast, mammalian cell cultures, and bacteria. (B) Growth of W303 yeast expressing various OtDUB fragments from the galactose-inducible promoter in p416GAL1. Yeast cultures were serially diluted in 10-fold steps and spotted on SD lacking uracil containing either galactose or glucose as the carbon source and grown for 3 d at 30 °C. (C) Venn diagram of total proteins identified between GST, OtDUB_275–1369, and OtDUB_675–1369. (D) Candidate interactors of indicated OtDUB fragments from LC-MS/MS. Total peptide counts are shown.

Fig. 2.

OtDUB binds Rac1 and Cdc42 and catalyzes nucleotide exchange in vitro. (A) Inputs and anti-Flag immunoprecipitates of lysates from HeLa cells ectopically expressing the indicated Flag-tagged OtDUB fragments. Proteins were resolved by SDS-PAGE and immunoblotted for Rho GTPases (Rac1,2,3; Cdc42; RhoA). (B) FLAG (Upper) and reciprocal GST (Lower) pulldown experiments between purified recombinant FLAG-tagged OtDUB fragments and GST-tagged Rac1 or Cdc42. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (C) Time course of the dissociation of BODIPY-GDP from Rac1 or Cdc42 in the presence of OtDUB_275–1369 (0, 50, or 250 nM) as measured by loss of BODIPY-GDP fluorescence. Excitation and emission wavelength were 488 nm and 535 nm, respectively.

Fig. 3.

A 200-residue OtDUB subdomain sufficient for binding Rac1. (A) OtDUB truncations used for mapping studies (Upper) and GST pulldown experiment using GST-OtDUB fragments as bait and Rac1 as prey (Lower). Gel was stained with Coomassie Blue. N-terminal truncation to residue 580 abolished binding, whereas the 548–759 fragment (OtDUB_GEF domain) retained full binding capacity. (B) SEC of OtDUB_GEF:Rac1 mixtures demonstrates stable complex formation (Upper). Column fractions were evaluated by SDS-PAGE and protein staining (Lower). (C) Time course of the dissociation of BODIPY-GDP from Rac1 in the presence of increasing amounts of OtDUB_GEF (residues 548–759). Raw fluorescence curves were fit to a single exponential decay (Upper), and initial rates were plotted against Rac1 concentration (Lower). Linear fit of the initial velocities yielded a k_cat/K_M of 2.6 ± 0.3 × 10⁵ M⁻¹·s⁻¹.

Fig. 4.

Crystal structure of the apo OtDUB_GEF domain reveals a unique topology. (A) Overall structural comparison of apo OtDUB_GEF and other Rho GEFs: Map (PDB ID code: 3GCG), SopE (PDB ID code: 1GZS), TIAM1 (PDB ID code: 1FOE). All structures shown with helices as cylinders and transparent surface. (B) Topology diagrams of the same GEFs color-ramped from N terminus (blue) to C terminus (red) to highlight the unique topology of OtDUB_GEF. Helices are shown as rectangles, beta strands are shown as arrows, and loop regions are shown as lines.

Fig. 5.

Biochemical function and structure of the OtDUB_GEF:Rac1 complex. (A) Three orthogonal views of the complex with OtDUB_GEF in purple and Rac1 in cyan. Switch I and II loops are in salmon and yellow, respectively, and the β2–3 hairpin selectivity interface is green. (B) Close-up views of each key interface between OtDUB_GEF and Rac1 showing selected residues, with hydrogen bonds and electrostatic interactions shown as dashes and water molecules as red spheres. (C) GST pulldown assays (Left) of GST-OtDUB_GEF (WT or charge-neutralizing mutations at each interface) incubated with Rac1 and analyzed by SDS/PAGE and Coomassie Blue staining. Corresponding BODIPY-GDP release assays are shown at Right.

Fig. 6.

OtDUB_GEF utilizes a unique carbonyl “catalytic lever” to catalyze nucleotide exchange. (A) Close-up views of switch II in GDP-bound Rac1 (gray, PDB ID code: 5N6O), three overlaid GEF-bound structures (slate is TIAM1:Rac1, PDB ID code: 1FOE; red is SopE:Cdc42, PDB ID code: 1GZS; and yellow is Map:Cdc42, PDB ID code: 3GCG), and the OtDUB_GEF-bound Rac1 structure (cyan). Unlike the three overlaid GEF-bound conformations, Glu⁶² in the OtDUB_GEF-bound structure is most similar to that found in GDP-bound Rac1. GEFs have been removed for clarity, and switch I residues are shown as sticks. BODIPY-GDP exchange assay reveals that Glu⁶² is not required for efficient exchange catalyzed by OtDUB_GEF. (B, Upper) A loop of OtDUB_GEF that interrupts α2 acts as a “catalytic lever” to promote release of GDP. OtDUB_GEF is shown as cartoon (purple), and Rac1 is shown as transparent surface (cyan). GDP (not present in our structure) is modeled based on PDB ID code 5N6O for reference. The steric clash and electrostatic repulsion are indicated by the red lines. (B, Lower) Nucleotide exchange assay reveals a strict requirement in the lever segment for exchange of BODIPY-GDP. (C) Calculated electrostatic surface potential map (unit k_BT/e) shows high negative charge in the OtDUB_GEF lever formed by a tricarbonyl motif at the vicinity of the diphosphate group of GDP.

Fig. 7.

OtDUB_GEF specifically activates Rac1 and modulates cell morphology. (A) Lysates from HeLa cells carrying empty vector or plasmids expressing WT or E572A OtDUB were subjected to GST-Pak1PBD pulldowns to enrich active Rac1 and Cdc42. Representative experiment (Upper) from four independent experiments that were quantified (Lower) relative to input levels and the empty vector control. Bars represent mean and SD P value determined from a two-tailed unpaired Student t test. Outlier from Rac1 wild type (5.4-fold increase in activity) excluded one trial. Outlier was identified using Grubbs algorithm with alpha = 0.05. (B) Representative epifluorescence images of fibroblasts carrying the vector expressing only GFP or plasmids expressing WT or E572A OtDUB_GEF. (C) Quantification of cell area, perimeter, and major axis length.