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Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins

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Fic proteins that are defined by the ubiquitous FIC (filamentation induced by cyclic AMP) domain are known to catalyse adenylylation (also called AMPylation); that is, the transfer of AMP onto a target protein. In mammalian cells, adenylylation of small GTPases through Fic proteins injected by pathogenic bacteria can cause collapse of the actin cytoskeleton and cell death1,2. It is unknown how this potentially deleterious adenylylation activity is regulated in the widespread Fic proteins that are found in all domains of life and that are thought to have critical roles in intrinsic signalling processes. Here we show that FIC-domain-mediated adenylylation is controlled by a conserved mechanism of ATP-binding-site obstruction that involves an inhibitory α-helix (αinh) with a conserved (S/T)XXXE(G/N) motif, and that in this mechanism the invariable glutamate competes with ATP γ-phosphate binding. Consistent with this, FIC-domain-mediated growth arrest of bacteria by the VbhT toxin of Bartonella schoenbuchensis is intermolecularly repressed by the VbhA antitoxin through tight binding of its αinh to the FIC domain of VbhT, as shown by structure and function analysis. Furthermore, structural comparisons with other bacterial Fic proteins, such as Fic of Neisseria meningitidis and of Shewanella oneidensis, show that αinh frequently constitutes an amino-terminal or carboxy-terminal extension to the FIC domain, respectively, partially obstructing the ATP binding site in an intramolecular manner. After mutation of the inhibitory motif in various Fic proteins, including the human homologue FICD (also known as HYPE), adenylylation activity is considerably boosted, consistent with the anticipated relief of inhibition. Structural homology modelling of all annotated Fic proteins indicates that inhibition by αinh is universal and conserved through evolution, as the inhibitory motif is present in 90% of all putatively adenylylation-active FIC domains, including examples from all domains of life and from viruses. Future studies should reveal how intrinsic or extrinsic factors modulate adenylylation activity by weakening the interaction of αinh with the FIC active site.

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Figure 1: The small protein VbhA represses the toxic effect (growth arrest) that is mediated by the adenylylation activity of VbhT in E. coli.
Figure 2: Structures and classification of Fic proteins according to the position of the inhibitory motif along the polypeptide chain.
Figure 3: Structure and function of wild-type and mutant NmFic reveals a general inhibition mechanism corroborated by HYPE protein analysis.

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Protein Data Bank

Data deposits

The atomic coordinates of VbhA–VbhT(FIC) and the complexes of NmFic, NmFic(SE/AA) and NmFic(D8) with AMPPNP have been deposited in the Protein Data Bank under accession codes 3SHG, 3S6A, 3SN9 and 3SE5, respectively.

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  • 25 January 2012

    The supplementary information PDF was replaced as Supplementary Figure 8 had corrupted in the original file posted on line.


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

    Article  CAS  Google Scholar 

  2. 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 

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

    Article  Google Scholar 

  4. 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)

    Article  CAS  Google Scholar 

  5. 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 

  6. Xiao, J., Worby, C. A., Mattoo, S., Sankaran, B. & Dixon, J. E. Structural basis of Fic-mediated adenylylation. Nature Struct. Mol. Biol. 17, 1004–1010 (2010)

    Article  CAS  Google Scholar 

  7. Dehio, C. et al. Bartonella schoenbuchii sp. nov., isolated from the blood of wild roe deer. Int. J. Syst. Evol. Microbiol. 51, 1557–1565 (2001)

    Article  CAS  Google Scholar 

  8. Engel, P. et al. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella . PLoS Genet. 7, e1001296 (2011)

    Article  CAS  Google Scholar 

  9. Schulein, R. et al. A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc. Natl Acad. Sci. USA 102, 856–861 (2005)

    Article  CAS  ADS  Google Scholar 

  10. 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)

    Article  CAS  Google Scholar 

  11. Utsumi, R., Nakamoto, Y., Kawamukai, M., Himeno, M. & Komano, T. Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant. J. Bacteriol. 151, 807–812 (1982)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Engelberg-Kulka, H., Amitai, S., Kolodkin-Gal, I. & Hazan, R. Bacterial programmed cell death and multicellular behavior in bacteria. PLoS Genet. 2, e135 (2006)

    Article  Google Scholar 

  13. 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)

    Article  CAS  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  ADS  Google Scholar 

  15. 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 

  16. Mukherjee, S. et al. Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature 477, 103–106 (2011)

    Article  CAS  ADS  Google Scholar 

  17. Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005)

    Article  Google Scholar 

  18. Self, A. J. & Hall, A. Purification of recombinant Rho/Rac/G25K from Escherichia coli . Methods Enzymol. 256, 3–10 (1995)

    Article  CAS  Google Scholar 

  19. Smith, S. J. & Rittinger, K. Preparation of GTPases for structural and biophysical analysis. Methods Mol. Biol. 189, 13–24 (2002)

    CAS  PubMed  Google Scholar 

  20. Leslie, A. G. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)

    Article  Google Scholar 

  21. Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  22. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  23. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  24. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  25. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  26. Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999)

    Article  CAS  Google Scholar 

  27. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010)

    Article  ADS  Google Scholar 

  28. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690 (2006)

    Article  CAS  Google Scholar 

  29. Schmidt, A. et al. Absolute quantification of microbial proteomes at different states by directed mass spectrometry. Mol. Syst. Biol. 7, 510 (2011)

    Article  Google Scholar 

  30. Zheng, L., Baumann, U. & Reymond, J. L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004)

    Article  Google Scholar 

  31. Dehio, C. & Meyer, M. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli . J. Bacteriol. 179, 538–540 (1997)

    Article  CAS  Google Scholar 

  32. Rhomberg, T. A., Truttmann, M. C., Guye, P., Ellner, Y. & Dehio, C. A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome. Cell. Microbiol. 11, 927–945 (2009)

    Article  CAS  Google Scholar 

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We thank T. Glatter for mass spectrometry analysis of samples at the Core Proteomics facility. We thank the staff of beamline X06SA of the Swiss Light Source for assistance with data acquisition. We are grateful to G. Pluschke for providing the genomic DNA of Neisseria meningitidis, the ASU Biodesign Institute for providing the plasmid enclosing the Shewanella oneidensis Fic protein and S. Mattoo and J. Dixon for providing the pET-GSTX plasmids enclosing HYPE and HYPE(H295A). We also thank D. Bumann and A. Boehm for providing plasmid pC10E and E. coli strain AB472, respectively. This work was supported by grants 3100-061777 and 3100-138414 from the Swiss National Science Foundation (to C.D. and T.S., respectively), and grant 51RT 0_126008 (InfectX) in the frame of the Swiss Initiative for Systems Biology (to C.D.).

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Authors and Affiliations



Author Contributions P.E., F.V.S. and A.H. cloned recombinant plasmids. P.E. discovered and physiologically characterized VbhT–VbhA as a toxin–antitoxin module and carried out the bioinformatic analysis. A.G. expressed, purified and crystallized VbhA–VbhT(FIC), NmFic(SE/AA) and NmFic(Δ8), and determined their structures. F.V.S. expressed, purified and crystallized NmFic with AMPPNP and determined the structure. A.G. and A.H. performed the adenylylation assays. A.H carried out the growth curve experiments. A.S. conducted the mass spectrometry analysis. All authors contributed to experimental design and data analysis. The manuscript was written by P.E., A.G., T.S. and C.D.

Corresponding authors

Correspondence to Tilman Schirmer or Christoph Dehio.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text (see Contents for details), Supplementary Figures 1-12 with legends, Supplementary Tables 1-3 and 5-6 (see separate file for Supplementary Table 4) and additional references. This was replaced on 25 January 2012 as Supplementary Figure 8 had corrupted in the original file posted on line. (PDF 12210 kb)

Supplementary Data 4

This file contains the classification of all analyzed PFAM FIC domain-containing proteins according to the presence of an anti-toxin (class I) or an intrinsic inhibition motif (class II and III). (XLS 1098 kb)

Supplementary Movie 1

This movie shows a model for the mechanism of active site obstruction in Fic proteins. (MOV 3573 kb)

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Engel, P., Goepfert, A., Stanger, F. et al. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. Nature 482, 107–110 (2012).

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