Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis of Fic-mediated adenylylation

Nature Structural & Molecular Biology volume 17, pages 1004–1010 (2010)Cite this article

Subjects

Abstract

The Fic family of adenylyltransferases, defined by a core HPFx(D/E)GN(G/K)R motif, consists of over 2,700 proteins found in organisms from bacteria to humans. The immunoglobulin-binding protein A (IbpA) from the bacterial pathogen Histophilus somni contains two Fic domains that adenylylate the switch1 tyrosine residue of Rho-family GTPases, allowing the bacteria to subvert host defenses. Here we present the structure of the second Fic domain of IbpA (IbpAFic2) in complex with its substrate, Cdc42. IbpAFic2-bound Cdc42 mimics the GDI-bound state of Rho GTPases, with both its switch1 and switch2 regions gripped by IbpAFic2. Mutations disrupting the IbpAFic2–Cdc42 interface impair adenylylation and cytotoxicity. Notably, the switch1 tyrosine of Cdc42 is adenylylated in the structure, providing the first structural view for this post-translational modification. We also show that the nucleotide-binding mechanism is conserved among Fic proteins and propose a catalytic mechanism for this recently discovered family of enzymes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ribbon representation of IbpAFic2 structure.
Figure 2: Crystal structure of the IbpAFic2 H3717A–Cdc42 complex.
Figure 3: IbpAFic2–Cdc42 binding interface.
Figure 4: Nucleotide binding by IbpAFic2.
Figure 5: IbpAFic2 mutants show reduced cytotoxicity.
Figure 6: A proposed catalytic mechanism of the Fic family proteins.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. http://www.ebi.ac.uk/interpro/IEntry?ac=IPR003812. InterPro: IPR003812 Filamentation induced by cAMP/death on curing, related. (2010).

  2. Worby, C.A. et al. The fic domain: regulation of cell signaling by adenylylation. Mol. Cell 34, 93–103 (2009).

    Article  CAS  Google Scholar 

  3. Yarbrough, M.L. et al. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269–272 (2009).

    Article  CAS  Google Scholar 

  4. Zekarias, B. et al. Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier. Infect. Immun. 78, 1850–1858 (2010).

    Article  CAS  Google Scholar 

  5. Lehnherr, H., Maguin, E., Jafri, S. & Yarmolinsky, M.B. Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. J. Mol. Biol. 233, 414–428 (1993).

    Article  CAS  Google Scholar 

  6. Liu, M., Zhang, Y., Inouye, M. & Woychik, N.A. Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit. Proc. Natl. Acad. Sci. USA 105, 5885–5890 (2008).

    Article  CAS  Google Scholar 

  7. Magnuson, R. & Yarmolinsky, M.B. Corepression of the P1 addiction operon by Phd and Doc. J. Bacteriol. 180, 6342–6351 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Roy, C.R. & Mukherjee, S. Bacterial FIC proteins AMP up infection. Sci. Signal. 2, pe14 (2009).

    Article  Google Scholar 

  9. Schmid, M.C. et al. A translocated bacterial protein protects vascular endothelial cells from apoptosis. PLoS Pathog. 2, e115 (2006).

    Article  Google Scholar 

  10. Xu, R.Q. et al. AvrAC(Xcc8004), a type III effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular tissues of Arabidopsis thaliana ecotype Col-0. J. Bacteriol. 190, 343–355 (2008).

    Article  CAS  Google Scholar 

  11. May, B.J. et al. Complete genomic sequence of Pasteurella multocida, Pm70. Proc. Natl. Acad. Sci. USA 98, 3460–3465 (2001).

    Article  CAS  Google Scholar 

  12. Lee, C.C. et al. Crystal structure of the type III effector AvrB from Pseudomonas syringae. Structure 12, 487–494 (2004).

    Article  CAS  Google Scholar 

  13. Desveaux, D. et al. Type III effector activation via nucleotide binding, phosphorylation, and host target interaction. PLoS Pathog. 3, e48 (2007).

    Article  Google Scholar 

  14. Kinch, L.N., Yarbrough, M.L., Orth, K. & Grishin, N.V. Fido, a novel AMPylation domain common to fic, doc, and AvrB. PLoS One 4, e5818 (2009).

    Article  Google Scholar 

  15. Stadtman, E.R., Shapiro, B.M., Kingdon, H.S., Woolfolk, C.A. & Hubbard, J.S. Cellular regulation of glutamine synthetase activity in Escherichia coli. Adv. Enzyme Regul. 6, 257–289 (1968).

    Article  CAS  Google Scholar 

  16. Brown, M.S., Segal, A. & Stadtman, E.R. Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the P II -regulatory protein. Proc. Natl. Acad. Sci. USA 68, 2949–2953 (1971).

    Article  CAS  Google Scholar 

  17. Hochstrasser, M. Origin and function of ubiquitin-like proteins. Nature 458, 422–429 (2009).

    Article  CAS  Google Scholar 

  18. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006).

    Article  CAS  Google Scholar 

  19. Shuman, S. & Lima, C.D. The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases. Curr. Opin. Struct. Biol. 14, 757–764 (2004).

    Article  CAS  Google Scholar 

  20. Shuman, S. DNA ligases: progress and prospects. J. Biol. Chem. 284, 17365–17369 (2009).

    Article  CAS  Google Scholar 

  21. Das, D. et al. Crystal structure of the Fic (filamentation induced by cAMP) family protein SO4266 (gi,24375750) from Shewanella oneidensis MR-1 at 1.6 Å resolution. Proteins 75, 264–271 (2009).

    Article  CAS  Google Scholar 

  22. Tagawa, Y. et al. Genetic and functional analysis of Haemophilus somnus high molecular weight-immunoglobulin binding proteins. Microb. Pathog. 39, 159–170 (2005).

    Article  CAS  Google Scholar 

  23. Scheffzek, K., Stephan, I., Jensen, O.N., Illenberger, D. & Gierschik, P. The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI. Nat. Struct. Biol. 7, 122–126 (2000).

    Article  CAS  Google Scholar 

  24. Hoffman, G.R., Nassar, N. & Cerione, R.A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345–356 (2000).

    Article  CAS  Google Scholar 

  25. Luong, P. et al. Kinetic and structural insights into the mechanism of AMPylation by VopS FIC domain. J. Biol. Chem. 285, 20155–20163 (2010).

    Article  CAS  Google Scholar 

  26. Hirsch, A.K., Fischer, F.R. & Diederich, F. Phosphate recognition in structural biology. Angew. Chem. Int. Edn Engl. 46, 338–352 (2007).

    Article  CAS  Google Scholar 

  27. Hoshinoo, K., Sasaki, K., Tanaka, A., Corbeil, L.B. & Tagawa, Y. Virulence attributes of Histophilus somni with a deletion mutation in the ibpA gene. Microb. Pathog. 46, 273–282 (2009).

    Article  CAS  Google Scholar 

  28. Prehna, G., Ivanov, M.I., Bliska, J.B. & Stebbins, C.E. Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors. Cell 126, 869–880 (2006).

    Article  CAS  Google Scholar 

  29. Galan, J.E. Common themes in the design and function of bacterial effectors. Cell Host Microbe 5, 571–579 (2009).

    Article  CAS  Google Scholar 

  30. Elde, N.C. & Malik, H.S. The evolutionary conundrum of pathogen mimicry. Nat. Rev. Microbiol. 7, 787–797 (2009).

    Article  CAS  Google Scholar 

  31. DerMardirossian, C. & Bokoch, G.M. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15, 356–363 (2005).

    Article  CAS  Google Scholar 

  32. Rossman, K.L. et al. A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange. EMBO J. 21, 1315–1326 (2002).

    Article  CAS  Google Scholar 

  33. Lammers, M., Meyer, S., Kuhlmann, D. & Wittinghofer, A. Specificity of interactions between mDia isoforms and Rho proteins. J. Biol. Chem. 283, 35236–35246 (2008).

    Article  CAS  Google Scholar 

  34. Yang, J., Zhang, Z., Roe, S.M., Marshall, C.J. & Barford, D. Activation of Rho GTPases by DOCK exchange factors is mediated by a nucleotide sensor. Science 325, 1398–1402 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the University of California, San Diego X-ray facility staff and the Advanced Light Source (beam line 8.2.1) staff of Lawrence Berkeley National Laboratory for beam access and help with data collection, Z. Xu and X. Guo for critically reading the manuscript and members of the Dixon laboratory for helpful discussions. This work was supported by US National Institutes of Health grant AI060662 to J.E.D.

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

    Junyu Xiao, Carolyn A Worby & Jack E Dixon

  2. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA

    Carolyn A Worby & Jack E Dixon

  3. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA

    Carolyn A Worby & Jack E Dixon

  4. Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, USA

    Seema Mattoo & Jack E Dixon

  5. Berkeley Center for Structural Biology, Lawrence Berkeley Laboratory, Berkeley, California, USA

    Banumathi Sankaran

Authors
  1. Junyu Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carolyn A Worby
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seema Mattoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Banumathi Sankaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jack E Dixon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.X. performed most of the research; S.M. purified VopS protein; B.S. collected diffraction data for IbpAFic2-alone crystals; J.X., C.A.W., S.M. and J.E.D. designed the research, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Jack E Dixon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Methods (PDF 737 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiao, J., Worby, C., Mattoo, S. et al. Structural basis of Fic-mediated adenylylation. Nat Struct Mol Biol 17, 1004–1010 (2010). https://doi.org/10.1038/nsmb.1867

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1867

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing