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The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

Nature Chemical Biology volume 9, pages 811–817 (2013)Cite this article

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Abstract

Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

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Figure 1: Doc inhibits translation by inactivation of ternary complex formation.
Figure 2: Doc phosphorylates EF-Tu at the conserved Thr382.
Figure 3: Inhibition of phosphorylation of EF-Tu and its dephosphorylation.
Figure 4: Solution structure of the Doc–EF-Tu–GDP complex by SAXS.
Figure 5: Chemical shift mapping of the nucleotide and EF-Tu binding sites of Doc.
Figure 6: Proposed catalytic mechanism of the Doc-type Fic kinases.

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Acknowledgements

We thank R. van Nues and D. Forrest for critical reading of the manuscript, A. Talavera for assistance with the docking experiments and J. Gray for support with MS. This work was supported by UK Biotechnology and Biological Sciences Research Council and the European Research Council (ERC-2007-StG 202994-MTP) to N.Z., the Onderzoeksraad of the VUB, Fonds Wetenschappelijk Onderzoek (FWO)-Vlaanderen, VIB and the Hercules Foundation. The authors acknowledge the use of the EMBL beamline P12 at the DESY synchrotron (Hamburg, Germany).

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Author notes

  1. Daniel Castro-Roa and Abel Garcia-Pino: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

    Daniel Castro-Roa & Nikolay Zenkin

  2. Department of Biotechnology, Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium

    Abel Garcia-Pino, Steven De Gieter, Nico A J van Nuland & Remy Loris

  3. Department of Structural Biology, Molecular Recognition Unit, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Brussels, Belgium

    Abel Garcia-Pino, Steven De Gieter, Nico A J van Nuland & Remy Loris

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  1. Daniel Castro-Roa
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Contributions

D.C.-R. performed biochemical experiments; S.D.G. prepared Doc and Phd samples; A.G.-P. prepared Doc and Phd samples, performed NMR and SAXS experiments, analyzed structural data and wrote the paper; N.A.J.v.N. performed NMR experiments; R.L. analyzed structural data and wrote the paper; N.Z. wrote the paper and supervised the project.

Corresponding authors

Correspondence to Abel Garcia-Pino or Nikolay Zenkin.

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Castro-Roa, D., Garcia-Pino, A., De Gieter, S. et al. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Nat Chem Biol 9, 811–817 (2013). https://doi.org/10.1038/nchembio.1364

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