Letter | Published:

Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination

Naturevolume 557pages734738 (2018) | Download Citation

Abstract

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins1. Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD+ binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277–PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Hershko, A., Ciechanover, A. & Varshavsky, A. The ubiquitin system. Nat. Med. 6, 1073–1081 (2000).

  2. 2.

    Qiu, J. et al. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533, 120–124 (2016).

  3. 3.

    Bhogaraju, S. et al. Phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination. Cell 167, 1636–1649.e13 (2016).

  4. 4.

    Wong, K., Kozlov, G., Zhang, Y. & Gehring, K. Structure of the Legionella effector, lpg1496, suggests a role in nucleotide metabolism. J. Biol. Chem. 290, 24727–24737 (2015).

  5. 5.

    Ji, X. et al. Mechanism of allosteric activation of SAMHD1 by dGTP. Nat. Struct. Mol. Biol. 20, 1304–1309 (2013).

  6. 6.

    Simon, N. C., Aktories, K. & Barbieri, J. T. Novel bacterial ADP-ribosylating toxins: structure and function. Nat. Rev. Microbiol. 12, 599–611 (2014).

  7. 7.

    Barrio, J. R., Secrist, J. A. III & Leonard, N. J. A fluorescent analog of nicotinamide adenine dinucleotide. Proc. Natl Acad. Sci. USA 69, 2039–2042 (1972).

  8. 8.

    Dong, Y. et al. Structural basis of ubiquitin modification by the Legionella effector SdeA. Nature https://doi.org/10.1038/s41586-018-0146-7 (2018).

  9. 9.

    Kotewicz, K. M. et al. A single Legionella effector catalyzes a multistep ubiquitination pathway to rearrange tubular endoplasmic reticulum for replication. Cell Host Microbe 21, 169–181 (2017).

  10. 10.

    Matte, A., Tari, L. W. & Delbaere, L. T. How do kinases transfer phosphoryl groups? Structure 6, 413–419 (1998).

  11. 11.

    Kee, J.-M. & Muir, T. W. Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family. ACS Chem. Biol. 7, 44–51 (2012).

  12. 12.

    Fuhs, S. R. et al. Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162, 198–210 (2015).

  13. 13.

    Lima, C. D., Klein, M. G. & Hendrickson, W. A. Structure-based analysis of catalysis and substrate definition in the HIT protein family. Science 278, 286–290 (1997).

  14. 14.

    Akturk, A. et al. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector. Nature https://doi.org/10.1038/s41586-018-0147-6 (2018).

  15. 15.

    Thomsen, M. C. F. & Nielsen, M. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res. 40, W281–W287 (2012).

  16. 16.

    Nielsen, M. & Andreatta, M. NNAlign: a platform to construct and evaluate artificial neural network models of receptor-ligand interactions. Nucleic Acids Res. 45, W344–W349 (2017).

  17. 17.

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

  18. 18.

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

  19. 19.

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).

  20. 20.

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

  21. 21.

    Bricogne, G. et al. BUSTER version 2.10.2 (United Kingdom Global Phasing Ltd., Cambridge, 2017).

  22. 22.

    Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212–1225 (2017).

  23. 23.

    Franke, D. & Svergun, D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346 (2009).

  24. 24.

    Svergun, D. I. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76, 2879–2886 (1999).

  25. 25.

    Svergun, D., Barberato, C. & Koch, M. H. J. IUCr. CRYSOL – a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).

  26. 26.

    Kozin, M. B. & Svergun, D. I. IUCr. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33–41 (2001).

  27. 27.

    Götze, M. et al. StavroX—a software for analyzing crosslinked products in protein interaction studies. J. Am. Soc. Mass Spectrom. 23, 76–87 (2012).

  28. 28.

    Berger, K. H. & Isberg, R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7, 7–19 (1993).

  29. 29.

    Xu, L. et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010).

  30. 30.

    Luo, Z.-Q. & Isberg, R. R. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc. Natl Acad. Sci. USA 101, 841–846 (2004).

Download references

Acknowledgements

We thank J. Vogel for the gift of anti-SidC serum, R. Prabu for help with total mass analysis of proteolysed SdeA, Ö. Yildiz for sharing synchrotron time, A. Chaikuad for initial construct design of SdeA, T. Hunter and C. Lima for advice on histidine intermediate protocols, T. Hanke for the PDE mechanism scheme and discussion, T. Colby and I. Matic for their help with identifying ubiquitination sites of RTN4B by LC-MS/MS and B. Schulman and D. Scott for crystallography advice. Swiss Light Source beamtime was part of proposal 20161958. We thank the staff of SLS for their assistance in data collection as well as E. Veshkova, S. Rodriguez Gomez, S. Jelenic and F. Miljkovic for technical assistance; K. Koch, D. Höller, V. Dötsch and S. Knapp for comments on the paper; and D. Svergun’s group at beamline P12, PETRA III, EMBL-DESY for SAXS data collection. This work was supported by iNEXT (PID:3515), the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 742720), the DFG-funded Cluster of Excellence ‘‘Macromolecular Complexes’’ (EXC115), the DFG-funded SPP 1580 program ‘‘Intracellular Compartments as Places of Pathogen-Host-Interactions’’ (I.D.) and the LOEWE program Ubiquitin Networks (Ub-Net) and the LOEWE Center for Gene and Cell Therapy Frankfurt (CGT), both funded by the State of Hesse/Germany. NIH-NIAID grant R01AI127465 supported Z.-Q.L. The work of S.B. is also funded by a Goethe University Nachwuchsforscher grant.

Reviewer information

Nature thanks K. Gehring and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Sissy Kalayil, Sagar Bhogaraju.

Affiliations

  1. Institute of Biochemistry II, Goethe University Frankfurt - Medical Faculty, University Hospital, Frankfurt am Main, Germany

    • Sissy Kalayil
    • , Sagar Bhogaraju
    • , Florian Bonn
    • , Donghyuk Shin
    • , Yaobin Liu
    • , Paolo Grumati
    •  & Ivan Dikic
  2. Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany

    • Sissy Kalayil
    • , Sagar Bhogaraju
    • , Donghyuk Shin
    • , Yaobin Liu
    •  & Ivan Dikic
  3. Purdue Institute of Immunology, Inflammation and Infectious Diseases and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    • Ninghai Gan
    •  & Zhao-Qing Luo
  4. Max Planck Institute of Biochemistry, Department of Structural Cell Biology, Martinsried, Germany

    • Jérôme Basquin

Authors

  1. Search for Sissy Kalayil in:

  2. Search for Sagar Bhogaraju in:

  3. Search for Florian Bonn in:

  4. Search for Donghyuk Shin in:

  5. Search for Yaobin Liu in:

  6. Search for Ninghai Gan in:

  7. Search for Jérôme Basquin in:

  8. Search for Paolo Grumati in:

  9. Search for Zhao-Qing Luo in:

  10. Search for Ivan Dikic in:

Contributions

S.K., S.B. and I.D. conceived the project. S.K. performed initial crystallization. S.K. and S.B. performed crystal optimization, structure solution, protein purification and biochemistry. J.B. contributed to crystallization and structure solution. F.B. performed mass spectrometry. D.S. performed SAXS and contributed to protein purification. Y.L. and P.G. performed experiments with SdeAFL. N.G. performed bacterial infection experiments. S.K., S.B., Z.-Q.L. and I.D. analysed the data. S.K., S.B. and I.D. wrote the manuscript. I.D. supervised the project.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ivan Dikic.

Extended data figures and tables

  1. Extended Data Fig. 1 Catalytic core—SdeA213–907.

    a, Limited proteolysis of SdeA fragment 193–998 and subsequent analysis of the fragments by Coomassie-stained SDS gel and total mass analysis by mass spectrometry. b, In vitro ubiquitination of Rab33b by SdeAFL and SdeA213–907. c, Left, scattering profile of SdeA213–907 with calculated scattering curve from crystal structure. Gunier region is shown in inset. Top right, ab initio bead model from DAMMIN superimposed with crystal structure and shown in two orientations. Bottom right, pair distance distribution plot and DAMMIN model fitting results. Experiments were repeated independently twice with similar results (a, b). For gel source data, see Supplementary Fig. 1.Source Data.

  2. Extended Data Fig. 2 Role of AHL in SdeA.

    a, ε-NAD+ hydrolysis assay in the presence of SdeA (SdeA213–907 or SdeA213–907ΔAHL) and ubiquitin (wild-type or R42A_R72A). b, c, In vitro ubiquitination assay with mutations in loops connecting AHL to PDE and the mART catalytic core in SdeA213–907 (b) and in SdeAFL (c). d, Substrate ubiquitination and ubiquitin modification by SdeAFL and SdeA213–907 in HEK293T cells. Abcam Ub and Cell Signalling Ub antibodies were used to monitor the levels of unmodified ubiquitin and total ubiquitin, respectively. e, Limited proteolysis analysis of various SdeA constructs. All experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1.Source Data.

  3. Extended Data Fig. 3 Characterization of the mART domain.

    a, Superimposition of the mART core of SdeA with that of NAD+ bound structure of iota toxin (PDB: 4H0Y) from Clostridium perfringens, which ADP-ribosylates actin of host cells. Residues in SdeA that are predicted to be important for NAD+ binding and hydrolysis are labelled. b, In vitro ubiquitination assay with NAD+ binding site mutants in the mART core of SdeA213–907. c, Residues at the interface between the mART core and AHL in proximal conformation (AHLprox). d, In vitro ubiquitination assays with the mutants of SdeA213–907 mART core-AHLprox interface residues indicated in c. e, Comparison of ε-NAD+ hydrolysis by SdeA213–907 and NAD+ binding site mutants and mutants disrupting the predicted mART core–AHLprox interaction. f, In vitro ubiquitination assays with the mutants of SdeAFL mART core–AHLprox interface residues indicated in c. Experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1.Source Data.

  4. Extended Data Fig. 4 Interaction between PDE and mART core.

    a, Details of interaction between SdeA PDE and mART core. Important residues mediating the interaction are indicated in the insets. b, c, Testing in vitro substrate ubiquitination and ubiquitin modification by PDE–mART core interaction mutants in SdeA213–907 (b) and SdeAFL (c). Experiments were repeated twice independently with similar results. For gel source data, see Supplementary Fig. 1.

  5. Extended Data Fig. 5 Histidine intermediate in SdeA catalysis.

    a, In vitro ubiquitination reactions by various SdeA213–907 PDE site histidine mutants probed by Coomassie-stained SDS–PAGE. b, High-energy HCD fragmentation was used to generate fragments of the peptide backbone. We could identify multiple fragments of SdeA275–284 and Ub34–48, to further validate the identity of the bridged active site. c, In vitro ubiquitination reaction by SdeA213–907(H407N) mutant using rhodamine-labelled ubiquitin. d, In vitro ubiquitination reaction using HA-tagged ubiquitin. e, In vitro ubiquitination reaction by SdeA213–907(H407N) without and with heating probed by phosphostain. f, In vitro ubiquitination reaction using HA–ubiquitin by various PDE mutants. These experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1.

  6. Extended Data Fig. 6 PDE domain catalytic site.

    a, In vitro Rab33b ubiquitination by SdeA PDE mutants. b, In vitro ubiquitination assays with PDE catalytic site mutants. c, In vitro Rab33b ubiquitination assays with GFP–SdeAFL PDE catalytic site mutants purified from HEK293T cells. These experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1.

  7. Extended Data Fig. 7 Substrate specificity of SdeA.

    a, b, Fragmentation spectra of the bridged peptide indicating RTN4B ubiquitination sites. These experiments were done once. c, Sequence motif of target serine sequences of SdeA as computed by Seq2Logo.

  8. Extended Data Fig. 8 Chemical inhibition of SdeA.

    a, Chemical structure of adenosine-5′-thio-monophosphate (5′-AMPS). b, 5′-AMPS-mediated inhibition of Rab33b PR-ubiquitination by SdeAFL, SdeA213–597 and SdeA213–907 in the presence of ADPR-Ub. c, 5′-AMPS-mediated inhibition of RTN4B PR-ubiquitination by SdeAFL. d, In vitro ubiquitination by SdeAFL in the presence of increasing concentrations of 5′-AMPS and AMP. e, Apparent inhibition constants (Ki) of AMP and 5′-AMPS against SdeAFL calculated from quantification of substrate ubiquitination shown in d. f, PDE domain of SdeC ubiquitinates Rab33b and RTN4B. g, Effect of 5′-AMPS on ubiquitination by SdeC PDE. These experiments were done twice independently with similar results. For gel source data, see Supplementary Fig. 1.Source Data.

  9. Extended Data Fig. 9 Effect of SdeA substrate-binding mutations in vivo.

    a, In vitro Rab33b ubiquitination assays with GFP–SdeAFL substrate-binding mutants purified from HEK293T cells. b, Expression and translocation of SdeA using wild-type and various mutant strains of Legionella. c, CFU fold change monitored in wild-type Legionella and ΔsidEs strain complemented with substrate ubiquitination defective mutant plasmids (n = 3 biological replicates). Data shown as mean ± s.e.m. d, Co-localization of Legionella-containing vacuoles and RTN4 networks in primary mouse macrophages. Experiments in a, b and d were repeated twice independently with similar results. For gel source data, see Supplementary Fig. 1.Source Data.

  10. Extended Data Fig. 10 Size-exclusion chromatography profile of SdeAFL.

    SdeAFL shows dimeric behaviour in size-exclusion chromatography column (Superdex 200 16/60). This experiment was repeated twice independently with similar results. For the inset, n = 1. MW, molecular weight. For gel source data, see Supplementary Fig. 1.Source Data.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Tables 1-3, Supplementary Text and Supplementary Figure 1. The Supplementary Text includes: a description of mART domain and interaction between mART and PDE domains as observed in the crystal structure of SdeA213-907; a description of an AMP-based low-affinity inhibitor, which affects substrate ubiquitination by targeting the PDE domains of SdeA and SdeC, and a detailed description of SdeA PDE domain mediated serine ubiquitination reaction scheme shown in Figure 2d

  2. Reporting Summary

Source Data

About this article

Publication history

Received

Accepted

Published

Issue Date

DOI

https://doi.org/10.1038/s41586-018-0145-8

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.