Structure and function of Fic proteins

Key Points

  • Proteins with FIC domains are widespread in pathogenic and non-pathogenic bacteria, where they are found with more than 60 different architectures, suggesting that some subfamilies have conserved regulatory functions.

  • Fic proteins use a wide variety of cofactors and protein substrates, but they exploit a very similar catalytic mechanism to carry out post-translational modifications through the addition of AMP, other NMPs, phosphocholine or phosphate to substrate proteins.

  • Fic proteins modify diverse target proteins, including small GTPases in animal cells, protein kinases in plants and elongation factor Tu in bacteria. These modifications impair cytoskeletal, trafficking, signalling or translational functions of the target cell.

  • The regulation of Fic proteins has only just begun to be investigated. Several Fic families are regulated through the obstruction of their active site by autoinhibition or by antitoxins, or through a phosphodiesterase that removes the post-translational modification. The extent to which each subfamily is regulated and the nature of the regulatory signals remain unknown.

  • A major area for future research is the elucidation of the physiological functions of Fic proteins produced by bacterial pathogens, as this could yield opportunities for developing novel antimicrobial compounds.

Abstract

Fic proteins are a family of proteins characterized by the presence of a conserved FIC domain that is involved in the modification of protein substrates by the addition of phosphate-containing compounds, including AMP and other nucleoside monophosphates, phosphocholine and phosphate. Fic proteins are widespread in bacteria, and various pathogenic species secrete Fic proteins as toxins that mediate post-translational modifications of host cell proteins, to interfere with cytoskeletal, trafficking, signalling or translation pathways in the host cell. In this Review, we discuss the current knowledge of the structure, function and regulation of Fic proteins and consider important areas for future research.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Functions of Fic proteins.
Figure 2: The structural diversity of Fic proteins.
Figure 3: Selection of cofactors by Fic proteins.
Figure 4: Selection of targets for post-translational modification by Fic proteins.
Figure 5: Regulation of Fic proteins.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Kawamukai, M., Matsuda, H., Fujii, W., Utsumi, R. & Komano, T. Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli. J. Bacteriol. 171, 4525–4529 (1989).

    CAS  Article  Google Scholar 

  2. 2

    Garcia-Pino, A. et al. Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation. J. Biol. Chem. 283, 30821–30827 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Arbing, M. A. et al. Crystal structures of Phd-Doc, HigA, and YeeU establish multiple evolutionary links between microbial growth-regulating toxin-antitoxin systems. Structure 18, 996–1010 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Engel, P. et al. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. Nature 482, 107–110 (2012). This study uncovers a mechanism of inhibition common to the VbhA–VbhT toxin–antitoxin module and Fic toxins, which involves the insertion of an inhibitory glutamate into the FIC active site.

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    Article  Google Scholar 

  7. 7

    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). The first analysis of the evolutionary relationship between Fic, Doc and AvrB proteins, which suggests that they function in PTMs.

    Article  Google Scholar 

  8. 8

    Garcia-Pino, A., Zenkin, N. & Loris, R. The many faces of Fic: structural and functional aspects of Fic enzymes. Trends Biochem. Sci. 39, 121–129 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Cruz, J. W. & Woychik, N. A. Teaching Fido new modiFICation tricks. PLoS Pathog. 10, e1004349 (2014).

    Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Worby, C. A. et al. The Fic domain: regulation of cell signaling by adenylylation. Mol. Cell 34, 93–103 (2009). References 10 and 11 are the first reports of the biochemical activity of Fic toxins as PTM enzymes. Both articles describe AMPylation of host RHO GTPases during infection and show that this impairs their regulation of the cytoskeleton.

    CAS  Article  Google Scholar 

  12. 12

    Pan, X., Luhrmann, A., Satoh, A., Laskowski-Arce, M. A. & Roy, C. R. Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320, 1651–1654 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Mukherjee, S. et al. Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature 477, 103–106 (2011). This paper identifies phosphocholination as a novel PTM carried out by a Fic toxin during infection.

    CAS  Article  Google Scholar 

  14. 14

    Bunney, T. D. et al. Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions. Structure 22, 1831–1843 (2014).

    CAS  Article  Google Scholar 

  15. 15

    Tan, Y., Arnold, R. J. & Luo, Z. Q. Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination. Proc. Natl Acad. Sci. USA 108, 21212–21217 (2011).

    CAS  Article  Google Scholar 

  16. 16

    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).

    CAS  Article  Google Scholar 

  17. 17

    Geertsema, R. S. et al. IbpA DR2 subunit immunization protects calves against Histophilus somni pneumonia. Vaccine 29, 4805–4812 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Cherfils, J. & Zeghouf, M. Chronicles of the GTPase switch. Nat. Chem. Biol. 7, 493–495 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Woolery, A. R., Yu, X., LaBaer, J. & Orth, K. AMPylation of Rho GTPases subverts multiple host signaling processes. J. Biol. Chem. 289, 32977–32988 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237–241 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Roy, C. R. & Mukherjee, S. Bacterial FIC proteins AMP up infection. Sci. Signal. 2, e14 (2009).

    Article  Google Scholar 

  22. 22

    Cherfils, J. & Zeghouf, M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269–309 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Goody, P. R. et al. Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins. EMBO J. 31, 1774–1784 (2012).

    CAS  Article  Google Scholar 

  24. 24

    Oesterlin, L. K., Goody, R. S. & Itzen, A. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor. Proc. Natl Acad. Sci. USA 109, 5621–5626 (2012). This study investigates how PTMs of RAB GTPases affect their biochemical response to host cell regulators.

    CAS  Article  Google Scholar 

  25. 25

    Gavriljuk, K., Itzen, A., Goody, R. S., Gerwert, K. & Kotting, C. Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy. Proc. Natl Acad. Sci. USA 110, 13380–13385 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Hardiman, C. A. & Roy, C. R. AMPylation is critical for Rab1 localization to vacuoles containing Legionella pneumophila. mBio 5, e01035-13 (2014).

    Article  Google Scholar 

  27. 27

    Mattoo, S. et al. Comparative analysis of Histophilus somni immunoglobulin-binding protein A (IbpA) with other Fic domain-containing enzymes reveals differences in substrate and nucleotide specificities. J. Biol. Chem. 286, 32834–32842 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Castro-Roa, D. et al. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Nat. Chem. Biol. 9, 811–817 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Cruz, J. W. et al. Doc toxin is a kinase that inactivates elongation factor Tu. J. Biol. Chem. 289, 7788–7798 (2014). References 28 and 29 demonstrate that the Doc component of the Doc–Phd toxin–antitoxin module is a kinase that inactivates EF-Tu to block translation.

    CAS  Article  Google Scholar 

  30. 30

    Kjeldgaard, M. & Nyborg, J. Refined structure of elongation factor EF-Tu from Escherichia coli. J. Mol. Biol. 223, 721–742 (1992).

    CAS  Article  Google Scholar 

  31. 31

    Berchtold, H. et al. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126–132 (1993).

    CAS  Article  Google Scholar 

  32. 32

    Helaine, S. et al. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343, 204–208 (2014). This study uncovers a novel function of the Doc–Phd module in favouring the appearance of Salmonella enterica subsp. enterica serovar Typhimurium persisters.

    CAS  Article  Google Scholar 

  33. 33

    Feng, F. et al. A Xanthomonas uridine 5′-monophosphate transferase inhibits plant immune kinases. Nature 485, 114–118 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Mackey, D., Holt, B. F. III, Wiig, A. & Dangl, J. L. RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108, 743–754 (2002).

    CAS  Article  Google Scholar 

  35. 35

    Chung, E. H. et al. Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants. Cell Host Microbe 9, 125–136 (2011).

    CAS  Article  Google Scholar 

  36. 36

    Palanivelu, D. V. et al. Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA. Protein Sci. 20, 492–499 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Pulliainen, A. T. et al. Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production. Proc. Natl Acad. Sci. USA 109, 9581–9586 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Siamer, S. & Dehio, C. New insights into the role of Bartonella effector proteins in pathogenesis. Curr. Opin. Microbiol. 23, 80–85 (2015).

    CAS  Article  Google Scholar 

  39. 39

    Xiao, J., Worby, C. A., Mattoo, S., Sankaran, B. & Dixon, J. E. Structural basis of Fic-mediated adenylylation. Nat. Struct. Mol. Biol. 17, 1004–1010 (2010). This report presents the first and still unique structure of an AMPylating Fic toxin bound to its GTPase substrate.

    CAS  Article  Google Scholar 

  40. 40

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

    CAS  Article  Google Scholar 

  41. 41

    Campanacci, V., Mukherjee, S., Roy, C. R. & Cherfils, J. Structure of the Legionella effector AnkX reveals the mechanism of phosphocholine transfer by the FIC domain. EMBO J. 32, 1469–1477 (2013). This investigation determines the structure of a phosphocholinating Fic toxin with bound CDP-choline, highlighting how Fic proteins recognize their cofactors.

    CAS  Article  Google Scholar 

  42. 42

    Rahman, M. et al. Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections. Nat. Neurosci. 15, 871–875 (2012).

    CAS  Article  Google Scholar 

  43. 43

    Ham, H. et al. Unfolded protein response-regulated Drosophila Fic (dFic) protein reversibly AMPylates BiP chaperone during endoplasmic reticulum homeostasis. J. Biol. Chem. 289, 36059–36069 (2014).

    CAS  Article  Google Scholar 

  44. 44

    Sanyal, A. et al. A novel link between Fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response. J. Biol. Chem. 290, 8482–8499 (2015).

    CAS  Article  Google Scholar 

  45. 45

    Goepfert, A., Stanger, F. V., Dehio, C. & Schirmer, T. Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold. PLoS ONE 8, e64901 (2013).

    CAS  Article  Google Scholar 

  46. 46

    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).

    CAS  Article  Google Scholar 

  47. 47

    Mishra, S. et al. Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis. Protein Expr. Purif. 86, 58–67 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Yamaguchi, Y., Park, J. H. & Inouye, M. Toxin–antitoxin systems in bacteria and archaea. Annu. Rev. Genet. 45, 61–79 (2011).

    CAS  Article  Google Scholar 

  49. 49

    Schoebel, S., Blankenfeldt, W., Goody, R. S. & Itzen, A. High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA. EMBO Rep. 11, 598–604 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Folly-Klan, M. et al. A novel membrane sensor controls the localization and ArfGEF activity of bacterial RalF. PLoS Pathog. 9, e1003747 (2013).

    Article  Google Scholar 

  51. 51

    Grammel, M., Luong, P., Orth, K. & Hang, H. C. A chemical reporter for protein AMPylation. J. Am. Chem. Soc. 133, 17103–17105 (2011).

    CAS  Article  Google Scholar 

  52. 52

    Yu, X. et al. Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays. Mol. Cell. Proteomics 13, 3164–3167 (2014).

    CAS  Article  Google Scholar 

  53. 53

    Pieles, K., Glatter, T., Harms, A., Schmidt, A. & Dehio, C. An experimental strategy for the identification of AMPylation targets from complex protein samples. Proteomics 14, 1048–1052 (2014).

    CAS  Article  Google Scholar 

  54. 54

    Hedberg, C. & Itzen, A. Molecular perspectives on protein adenylylation. ACS Chem. Biol. 10, 12–21 (2015).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank the members of their laboratories and all of their colleagues from other laboratories whose research is described in this Review. This work was funded by the French Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche and the Fondation pour la Recherche Médicale (J.C.).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Craig R. Roy or Jacqueline Cherfils.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

PowerPoint slides

Glossary

Filamentation

A bacterial growth mode in which cells grow without division, resulting in a filamentous morphology. This type of growth occurs in response to various stresses.

Secretion systems

Protein nanomachines that deliver DNA and bacterially encoded effector proteins into target cells.

Small GTPases

A superfamily of GTP-binding proteins that are regulated by GDP and GTP, and by changing localization to the membrane or the cytosol. In turn, these GTPases regulate a variety of processes in eukaryotic cells, including cell growth and differentiation, dynamics of the actin cytoskeleton and membrane traffic. RAB and RHO small GTPases carry a prenyl lipid in their carboxyl terminus, and this moiety is necessary for their reversible attachment to membranes.

Cofactors

Chemical compounds that participate in the reaction catalysed by an enzyme.

Switch regions

Pertaining to small GTPases: two flexible regions that are found in all small GTPases; these regions change conformation on binding to GTP and are directly involved in the interactions of GTPases with regulators and effectors.

Inflammasome

A multiprotein oligomer that activates an inflammatory cascade during an infection.

Guanine nucleotide exchange factors

(GEFs). Activators of small GTPases. GEFs function by stimulating GDP–GTP exchange, and they often have membrane-binding domains that increase their activities by colocalizing them with their cognate small GTPases.

Guanine nucleotide dissociation inhibitors

(GDIs). Negative regulators of RHO- and RAB-family small GTPases. GDIs inactivate their targets by displacing them from membranes, forming a cytosolic complex with the small GTPases by masking their prenyl group.

Elongation factor Tu

(EF-Tu). A prokaryotic elongation factor that binds tRNA molecules and shuttles them to a free site on the ribosome. EF-Tu is a GTP-binding protein related to small GTPases, and it undergoes large conformational changes on activation by GDP–GTP exchange and on inactivation by GTP hydrolysis.

Persisters

Non-replicating cells that are present in low numbers in bacterial populations and show increased survival under a variety of environmental stresses, including antibiotic treatment.

Glia cells

Non-neuronal brain cells that regulate brain homeostasis.

Unfolded-protein response

A cellular stress response that is activated by the accumulation of misfolded proteins in the endoplasmic reticulum.

Nucleophilic attack

The donation of electrons by one electron-rich atom (the nucleophile) to another, electron-poor atom (the electrophile) to form a new chemical bond during enzymatic catalysis.

β-sheet augmentation

A mode of protein–protein interaction in which a β-strand from the ligand pairs with the edge of a preformed β-sheet of the acceptor protein.

Structural-genomics consortium

A large-scale initiative to determine protein structures and make them immediately available to the scientific community, often before anything is known about the function of the protein. About half the structures of Fic proteins have been determined by such structural-genomics centres.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Roy, C., Cherfils, J. Structure and function of Fic proteins. Nat Rev Microbiol 13, 631–640 (2015). https://doi.org/10.1038/nrmicro3520

Download citation

Further reading