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Comparative Study
. 2011 Sep 16;286(37):32834-42.
doi: 10.1074/jbc.M111.227603. Epub 2011 Jul 27.

Comparative Analysis of Histophilus Somni Immunoglobulin-Binding Protein A (IbpA) With Other Fic Domain-Containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities

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Free PMC article
Comparative Study

Comparative Analysis of Histophilus Somni Immunoglobulin-Binding Protein A (IbpA) With Other Fic Domain-Containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities

Seema Mattoo et al. J Biol Chem. .
Free PMC article

Abstract

A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.

Figures

FIGURE 1.
FIGURE 1.
Substrate specificity of IbpA-Fic2. A, the Fic domains of IbpA, PfhB2 and HYPE target Tyr-32 of Cdc42, whereas VopS targets Thr-35. Bacterially expressed GST-tagged IbpA-Fic2, IbpA-Fic1, PfhB2-Fic2, VopS, or HYPE-Fic was incubated with wild type (W), Y32F (Y) or T35A (T) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro adenylylation assay using [α-32P]ATP. Samples were separated on SDS-PAGE and visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). The position of Cdc42 on the gel is indicated by arrows. The Fic domains of IbpA, PfhB2, and HYPE adenylylate wild type Cdc42 and Cdc42-T35A but not Cdc42-Y32F, indicating their specificity for the switch 1 tyrosine. In contrast, VopS fails to adenylylate only Cdc42-T35A, indicating its specificity for the switch 1 threonine. B, IbpA-Fic2 targets both the active and the inactive forms of Rho GTPases. Bacterially expressed untagged Cdc42, Rac, and RhoA loaded with GDP or GMP-PNP (as described under “Experimental Procedures”) were incubated with IbpA-Fic2 in an in vitro adenylylation assay. The protein load was visualized by Coomassie Blue staining, and the amount of adenylylation was visualized by autoradiography. The nucleotide status of the GTPases was confirmed prior to adenylylation by incubation with GST-Pak (Cdc42 and Rac) or GST-Rhotekin (RhoA) followed by separation on SDS-PAGE and Western analysis using antibodies directed against the individual GTPases. C, IbpA-Fic2 is active against the Cdc42-RhoGDI complex. HA-tagged Cdc42 was expressed in HEK293A cells. Bacterially expressed His6-SUMO-RhoGDI bound to nickel-agarose beads was incubated with the HEK239A cell extract treated for GDP loading of Rho GTPases (as described under “Experimental Procedures.”) to allow Cdc42-RhoGDI complex formation. After washing, the beads were subjected to the in vitro adenylylation reaction in the presence or absence of GST-tagged IbpA-Fic2. The supernatant (supe) and bead eluate were separated on SDS-PAGE and visualized by autoradiography. The protein load was monitored by Ponceau S staining.
FIGURE 2.
FIGURE 2.
Apparent steady-state kinetic measurements for ATP and constitutively active Cdc42. A, initial velocity measurements for ATP were obtained using a constant concentration of Cdc421–179Q61L of 500 μm while varying the ATP concentrations from 100 to 10,000 μm. B, initial velocity measurements for Cdc42 were obtained at 5 mm ATP while varying the concentration of Cdc421–179Q61L between 100 and 2800 μm. Assays were performed in triplicate with IbpA-Fic2 at 0.56 nm. The line represents the fit of this data using the Michaelis-Menten equation (“Experimental Procedures”). Error bars indicate S.E.
FIGURE 3.
FIGURE 3.
Fic domains of IbpA, PfhB2, and VopS preferentially target the Rho subfamily of GTPases for adenylylation. A, survey of Ras family Rho GTPases as substrates for Fic-mediated adenylylation. The indicated GST-tagged Rho GTPases were bacterially expressed and purified and incubated with purified IbpA-Fic2 in an in vitro adenylylation reaction. Samples were separated on SDS-PAGE and visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). The position of IbpA-Fic2 on the gel is indicated by an arrow. IbpA-Fic2 adenylylated only the Rho family members, RhoB, RhoC, RhoG, and TC10. B, the ability of Fic enzymes to adenylylate RhoG. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-RhoG in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). The position of RhoG on the gel is indicated by an arrow. The Fic domains of IbpA, PfhB2, and VopS efficiently adenylylate RhoG, whereas the Fic domain of HYPE displays a weaker adenylylation activity. C, the ability of Fic enzymes to adenylylate TC10. GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, VopS, and HYPE-Fic were incubated with bacterially expressed and purified GST-TC10 in an in vitro adenylylation reaction. Samples separated by SDS-PAGE were visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). The position of TC10 on the gel is indicated by an arrow. The Fic domains of IbpA and PfhB2 can efficiently adenylylate TC10, whereas VopS shows minimal activity toward it. HYPE did not adenylylate TC10 in vitro.
FIGURE 4.
FIGURE 4.
Nucleotide specificity of IbpA-Fic2. A, GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His6-SUMO-tagged HYPE-Fic were incubated with Cdc421–179Q61L in an in vitro reaction using [α-32P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B, reactions with His6-SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography (upper panel) and Coomassie Blue staining (bottom panel). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C, point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α-32P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography (top panel) and Coomassie Blue staining (bottom panel). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D, comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type (W), Y32F (Y), or T35A (T) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α-32P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography (top panel) with exposure times adjusted for optimal visualization and by Coomassie Blue staining (lower panel). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.
FIGURE 5.
FIGURE 5.
Phylogenetic comparison of Fic enzymes with other classes of adenylyltransferases. A phylogenetic tree was generated using the neighbor joining method for each of the ATase families using the adenylyltransferase domain of index proteins shown in supplemental Table 1. The four families of ATases are shown: DNA/RNA ligases (in yellow) with a bracket indicating NAD+-specific enzymes; the GS-ATase family (in orange); the E1 ubiquitin ligase family (in chocolate brown); and the FiDo family (in red) with brackets indicating the HYPE, Doc, and Fic subgroups. NDL, NAD+ dependent ligase; ADL, ATP dependent ligase; RL, RNA ligase; KN, kanamycin nucleotidyltransferase.

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