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

Nature volume 482, pages 107–110 (2012)Cite this article

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Abstract

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.

Change history

  • 25 January 2012

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

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Acknowledgements

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 SystemsX.ch Swiss Initiative for Systems Biology (to C.D.).

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Author notes

  1. Philipp Engel

    Present address: Present address: Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520-8106, USA.,

  2. Philipp Engel and Arnaud Goepfert: These authors contributed equally to this work.

Authors and Affiliations

  1. Focal Area Infection Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland ,

    Philipp Engel, Arnaud Goepfert, Frédéric V. Stanger, Alexander Harms & Christoph Dehio

  2. Core Program Structural Biology and Biophysics, Biozentrum, University of Basel, CH-4056 Basel, Switzerland ,

    Arnaud Goepfert, Frédéric V. Stanger & Tilman Schirmer

  3. Proteomics Core Facility, Biozentrum, University of Basel, CH-4056 Basel, Switzerland ,

    Alexander Schmidt

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Contributions

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). https://doi.org/10.1038/nature10729

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