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Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture


Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

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Fig. 1: Schematic representation of the co-substrate-mediated covalent capture concept.
Fig. 2: IbpA1 binary probe reaction and subsequent covalent ternary complex formation with Cdc42.
Fig. 3: In cellulo covalent ternary complex formation.
Fig. 4: Crystal structure of covalently linked IbpA1Cdc42 complex and Fic enzyme alignment.
Fig. 5: Identification and validation of cellular targets for IbpA, BepA and HYPE.

Data availability

All relevant data are available from the corresponding authors upon reasonable request. The crystallography data and corresponding atomic model have been deposited in the PDB under accession code 6SIU. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium ( via the PRIDE53 partner repository, with data set identifier PXD015599. Data for Figs. 2 and 5 are available as source data with this paper.


  1. 1.

    Veyron, S., Peyroche, G. & Cherfils, J. FIC proteins: from bacteria to humans and back again. Pathog. Dis. 76, fty012 (2018).

    Google Scholar 

  2. 2.

    Casey, A. K. & Orth, K. Enzymes involved in AMPylation and deAMPylation. Chem. Rev. 118, 1199–1215 (2018).

    CAS  PubMed  Google Scholar 

  3. 3.

    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 

  4. 4.

    Kawamukai, M. et al. Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain. J. Bacteriol. 170, 3864–3869 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Komano, T., Utsumi, R. & Kawamukai, M. Functional analysis of the fic gene involved in regulation of cell division. Res. Microbiol. 142, 269–277 (1991).

    CAS  PubMed  Google Scholar 

  6. 6.

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

    CAS  PubMed  Google Scholar 

  7. 7.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Müller, M. P. et al. The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b. Science 329, 946–949 (2010).

    PubMed  Google Scholar 

  9. 9.

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

    CAS  PubMed  Google Scholar 

  10. 10.

    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, 3164–3176 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Lewallen, D. M., Steckler, C. J., Knuckley, B., Chalmers, M. J. & Thompson, P. R. Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates. Mol. Biosyst. 8, 1701–1706 (2012).

    CAS  PubMed  Google Scholar 

  12. 12.

    Smit, C. et al. Efficient synthesis and applications of peptides containing adenylylated tyrosine residues. Angew. Chem. Int. Ed. 50, 9200–9204 (2011).

    CAS  Google Scholar 

  13. 13.

    Albers, M. F., van Vliet, B. & Hedberg, C. Amino acid building blocks for efficient fmoc solid-phase synthesis of peptides adenylylated at serine or threonine. Org. Lett. 13, 6014–6017 (2011).

    CAS  PubMed  Google Scholar 

  14. 14.

    Niphakis, M. J. & Cravatt, B. F. Enzyme inhibitor discovery by activity-based protein profiling. Annu. Rev. Biochem. 83, 341–377 (2014).

    CAS  PubMed  Google Scholar 

  15. 15.

    Hu, C. W. et al. Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase. Nat. Chem. Biol. 13, 1267–1273 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Brauckhoff, N., Hahne, G., Yeh, J. T. H. & Grossmann, T. N. Protein-templated peptide ligation. Angew. Chem. Int. Ed. 53, 4337–4340 (2014).

    CAS  Google Scholar 

  18. 18.

    Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. & Koike, T. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol. Cell. Proteomics 5, 749–757 (2006).

    CAS  PubMed  Google Scholar 

  19. 19.

    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  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Wang, Z. Comprehensive Organic Name Reactions and Reagents (Wiley, 2009).

  21. 21.

    Perni, M. et al. Delivery of native proteins into C-elegans using a transduction protocol based on lipid vesicles. Sci. Rep. 7, 15045 (2017).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    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).

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47, W636–W641 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Broncel, M., Serwa, R. A., Bunney, T. D., Katan, M. & Tate, E. W. Global profiling of Huntingtin-associated protein E (HYPE)-mediated AMPylation through a chemical proteomic approach. Mol. Cell. Proteomics 15, 715–725 (2016).

    CAS  PubMed  Google Scholar 

  25. 25.

    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  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Preissler, S. et al. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum. eLife 4, e12621 (2015).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Topolska-Wos, A. M., Chazin, W. J. & Filipek, A. CacyBP/SIP — structure and variety of functions. Biochim. Biophys. Acta 1860, 79–85 (2016).

    CAS  PubMed  Google Scholar 

  28. 28.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Kielkowski, P. et al. FICD activity and AMPylation remodelling modulate human neurogenesis. Nat. Commun. 11, 517 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Sreelatha, A. et al. Protein AMPylation by an evolutionarily conserved pseudokinase. Cell 175, 809–821 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

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

    CAS  PubMed  Google Scholar 

  32. 32.

    Zwirzitz, A. et al. Lactoferrin is a natural inhibitor of plasminogen activation. J. Biol. Chem. 293, 8600–8613 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Ghosh, P., Dahms, N. M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nat. Rev. Mol. Cell Biol. 4, 202–212 (2003).

    CAS  PubMed  Google Scholar 

  34. 34.

    Kounnas, M. Z. et al. The α2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes pseudomonas exotoxin A. J. Biol. Chem. 267, 12420–12423 (1992).

    CAS  PubMed  Google Scholar 

  35. 35.

    Truttmann, M. C. et al. Bartonella henselae engages inside-out and outside-in signaling by integrin β1 and talin1 during invasome-mediated bacterial uptake. J. Cell Sci. 124, 3591–3602 (2011).

    CAS  PubMed  Google Scholar 

  36. 36.

    Weidow, C. L., Black, D. S., Bliska, J. B. & Bouton, A. H. CAS/Crk signalling mediates uptake of Yersinia into human epithelial cells. Cell. Microbiol. 2, 549–560 (2000).

    CAS  PubMed  Google Scholar 

  37. 37.

    Preissler, S. & Ron, D. Early events in the endoplasmic reticulum unfolded protein response. Cold Spring Harb. Perspect. Biol. 11, a033894 (2019).

    CAS  PubMed  Google Scholar 

  38. 38.

    Truttmann, M. C. et al. The caenorhabditis elegans protein FIC-1 is an AMPylase that covalently modifies heat-shock 70 family proteins, translation elongation factors and histones. PLoS Genet. 12, e1006023 (2016).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Chaudhary, S., Pak, J. E., Gruswitz, F., Sharma, V. & Stroud, R. M. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat. Protoc. 7, 453–466 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zhang, Z. & Marshall, A. G. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 9, 225–233 (1998).

    CAS  PubMed  Google Scholar 

  41. 41.

    Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  PubMed  Google Scholar 

  42. 42.

    Vettermann, C., Jack, H. M. & Mielenz, D. A colloidal silver staining–destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns. Anal. Biochem. 308, 381–387 (2002).

    CAS  PubMed  Google Scholar 

  43. 43.

    Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinform. 18, 529 (2017).

    Google Scholar 

  44. 44.

    Winter, G. et al. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr. D 74, 85–97 (2018).

    CAS  Google Scholar 

  45. 45.

    Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011).

    CAS  PubMed  Google Scholar 

  46. 46.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  Google Scholar 

  47. 47.

    Tickle, I. J. et al. STARANISO (2018).

  48. 48.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  Google Scholar 

  50. 50.

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

    CAS  PubMed  Google Scholar 

  51. 51.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  52. 52.

    Johnson, G. T., Autin, L., Goodsell, D. S., Sanner, M. F. & Olson, A. J. ePMV embeds molecular modeling into professional animation software environments. Structure 19, 293–303 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

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The synchrotron MX data were collected at beamline P14 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg). We thank G. Pompidor for the assistance in using the beamline. We also acknowledge technical support from the SPC facility at EMBL Hamburg. A.I. acknowledges access to the core facilities and laboratories of the Centre for Structural Systems Biology (CSSB, Hamburg). We thank M. Feige for allowing us to use the cell biology facility and C. Rulofs for general technical assistance as well as G. Schroeder for sharing the LtpG gene construct. B.G. was funded by a Technical University Foundation Fellowship (TUFF, 2016–2017) and an Alexander von Humboldt Post-doctoral Fellowship (AvH, 2017–2019). C.H. thanks the Knut and Alice Wallenberg Foundation Sweden (KAW 2013.0187) and the Swedish Research Council (VR) for generous support. The authors acknowledge the German Research Foundation for support through the Collaborative Research Centre SFB1035 (project B05).

Author information




B.G., C.H. and A.I. conceived and developed the concept and design, analysed the data and wrote the manuscript. B.G. performed the experiments and prepared the figures. J.F. performed the HYPE experiments and participated in manuscript writing. C.H. and A.I. designed and supervised the synthesis of the TReNDs. M.R., M.F.A. and C.P. synthesized the TReNDs. M.R. further optimized the TReND synthesis. C.K. performed the tryptic digestion and the LC-MS/MS experiments and analysed the data. H.S. supervised the LS-MS/MS experiments. V.P. and B.G. collected the X-ray diffraction data. V.P. analysed the X-ray data and refined the structure. A.I and C.H. oversaw the project, data analysis and data interpretation. All authors participated in manuscript editing and final approval.

Corresponding authors

Correspondence to Christian Hedberg or Aymelt Itzen.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Schematic representation of the co-substrate-mediated covalent capture workflow for target identification.

Fic enzymes with affinity tags are used to capture their putative targets in mammalian lysates by forming a covalent ternary complex. Those covalent ternary complexes are then enriched by affinity methods and subsequently subjected to tryptic digestion and LC-MS/MS for target identification.

Extended Data Fig. 2 Intact protein mass spectrometry demonstrating regioselectivity of TReNDs.

LC/ESI-MS measurements of endogenous cysteine containing proteins such as a, Legionella pneumophila effector DrrA/SidM b, human ras family GTPase HRas and c, human rab family GTPase Rab32 incubated with TReNDs. TReND mix indicates the mixture of TReND-1,2,3,4 and 5 in equimolar ratio. No unspecific coupling observed as shown by the deconvoluted mass spectrum upon TReND incubation. For all panels: Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Extended Data Fig. 3 Intact protein mass spectrometry demonstrating TReND-6 reactivity.

LC/ESI-MS measurements of Twin-Strep® tagged IbpA I3755C (TS-IbpA) and reactivity of β-γ non-hydrolysable TReND-6 with TS-IbpA. The mass increase of 544 Da reveals that TReND-6 coupled to IbpA and is resistant to hydrolysis even in the presence of 1 mM MgCl2. Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Extended Data Fig. 4 Composite OMIT map of IbpATReNDCdc42 X-ray crystal structure.

Electron density map covering the whole structure. b, Unbiased electron density map of TReND-1 as covalent linker. Electron density of phosphate atoms suggests an alternative conformation for the TReND-1 in the covalent ternary complex. The absence of adenine base allows the phosphate group and linked tyrosine 32 of Cdc42 to fill in space and adopt an alternative conformation, which is indicated as pink carbon atoms. Canonical conformation is indicated as light-green carbon atom. Electron density of tyrosine 32 is less resolved due to flexibility. OMIT map is constructed with sigma 1.0 and carve distance of 2 Å to the linker.

Extended Data Fig. 5 Intact protein mass spectrometry demonstrating TReND reactivity with other Fic enzymes.

LC/ESI-MS measurements of strategically placed cysteine mutants of Fic enzymes such as a, Bartonella hanselea BepA b, human HYPE/FicD c, Legionella pneumophila LpFic and d, Legionella pneumophila LtpG incubated with TReNDs. Corresponding cysteine modification and reactive TReND is indicated. For all panels: Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Extended Data Fig. 6 Peptide LC-MS/MS spectrum for AMPylation.

a, CACYBP AMPylation spectrum reveals Y71 as the modification site by IbpA. The experiment was repeated independently at least twice with similar results. b, EEF1A2 AMPylation spectrum reveals T432 and c, T261 as modification sites by HYPE/FicD. The experiments in the sections b) and c) were the confirmation of previously published results, therefore performed once.

Extended Data Fig. 7 IbpA:Cdc42 ternary complex with various TReNDs.

SDS-PAGE shows that only IbpA I3755C and (1) yielded a ternary complex with Cdc42. Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Extended Data Fig. 8 BepA cysteine mutants and their reactivity with various TReNDs.

PhosTag SDS-PAGE shows that only BepA S198C and N111C mutants reacted with TReNDs. BepA S198C mutant showed better reactivity towards (1) and lesser reactivity with (3). BepA N111C showed overall good reactivity with all the TReNDs. Supported with the MS data in Supplementary Fig. 5a, BepA N111C was chosen as the optimal cysteine substitution mutant. Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Extended Data Fig. 9 HYPE/FicD cysteine mutants and their reactivity with various TReNDs.

HYPE H319C mutant showed reactivity with TReNDs 1-3 in a certain degree. However, HYPE E404C showed greater reactivity with TReNDs 1-3. HYPE E404C and (1) was chosen as binary probe for subsequent ternary complex analysis. Whenever a representative result is shown, each experiment was repeated independently at least twice with similar results.

Supplementary information

Supplementary Information

Supplementary Note and Tables 1–4.

Reporting Summary

Supplementary Data 1

Tabulated MaxQuant output for mass spectrometry data.

Source data

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 5

Unprocessed gels and Western Blots.

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Gulen, B., Rosselin, M., Fauser, J. et al. Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture. Nat. Chem. 12, 732–739 (2020).

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