Abstract
The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2,3,4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA—a member of the SidE family—at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ–CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which—in the absence of glutamate or modifiable SdeA—undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Qiu, J. & Luo, Z. Q. Legionella and Coxiella effectors: strength in diversity and activity. Nat. Rev. Microbiol. 15, 591–605 (2017).
Qiu, J. et al. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533, 120–124 (2016).
Bhogaraju, S. et al. Phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination. Cell 167, 1636–1649 (2016).
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).
Liu, Y. & Luo, Z. Q. The Legionella pneumophila effector SidJ is required for efficient recruitment of endoplasmic reticulum proteins to the bacterial phagosome. Infect. Immun. 75, 592–603 (2007).
Jeong, K. C., Sexton, J. A. & Vogel, J. P. Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ. PLoS Pathog. 11, e1004695 (2015).
Qiu, J. et al. A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination. Cell Res. 27, 865–881 (2017).
Lin, Y. H. & Machner, M. P. Exploitation of the host cell ubiquitin machinery by microbial effector proteins. J. Cell Sci. 130, 1985–1996 (2017).
Song, L. & Luo, Z. Q. Post-translational regulation of ubiquitin signaling. J. Cell Biol. 218, 1776–1786 (2019).
Dorer, M. S., Kirton, D., Bader, J. S. & Isberg, R. R. RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics. PLoS Pathog. 2, e34 (2006).
Kalayil, S. et al. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination. Nature 557, 734–738 (2018).
Eddé, B. et al. Posttranslational glutamylation of alpha-tubulin. Science 247, 83–85 (1990).
Finn, R. D. et al. The Pfam protein families database. Nucleic Acids Res. 38, D211–D222 (2010).
Heidtman, M., Chen, E. J., Moy, M. Y. & Isberg, R. R. Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways. Cell. Microbiol. 11, 230–248 (2009).
Rhoads, A. R. & Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340 (1997).
Black, M. H. et al. Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases. Science 364, 787–792 (2019).
Van Petegem, F., Chatelain, F. C. & Minor, D. L., Jr. Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex. Nat. Struct. Mol. Biol. 12, 1108–1115 (2005).
Bagshaw, C. ATP analogues at a glance. J. Cell Sci. 114, 459–460 (2001).
Casey, A. K. & Orth, K. Enzymes involved in AMPylation and deAMPylation. Chem. Rev. 118, 1199–1215 (2018).
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).
O’Hagan, R. et al. Glutamylation regulates transport, specializes function, and sculpts the structure of cilia. Curr. Biol. 27, 3430–3441 (2017).
Drum, C. L. et al. Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415, 396–402 (2002).
Guo, Q. et al. Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin. EMBO J. 24, 3190–3201 (2005).
Gancedo, J. M. Biological roles of cAMP: variations on a theme in the different kingdoms of life. Biol. Rev. Camb. Philos. Soc. 88, 645–668 (2013).
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).
Luo, Z. Q. & Farrand, S. K. Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity. Proc. Natl Acad. Sci. USA 96, 9009–9014 (1999).
Xu, L. et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010).
Sheedlo, M. J. et al. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc. Natl Acad. Sci. USA 112, 15090–15095 (2015).
Mumberg, D., Müller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995).
Otwinowski, Z. & Minor, W. [20] Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).
Gan, N., Nakayasu, E. S., Hollenbeck, P. J. & Luo, Z. Q. Legionella pneumophila inhibits immune signalling via MavC-mediated transglutaminase-induced ubiquitination of UBE2N. Nat. Microbiol. 4, 134–143 (2019).
Mayampurath, A. M. et al. DeconMSn: a software tool for accurate parent ion monoisotopic mass determination for tandem mass spectra. Bioinformatics 24, 1021–1023 (2008).
Na, S., Bandeira, N. & Paek, E. Fast multi-blind modification search through tandem mass spectrometry. Mol. Cell. Proteomics 11, M111.010199 (2012).
Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).
Acknowledgements
We thank C. Fan for assistance with structure determination and for discussions, and K. Weitz for assistance with the mass spectrometry analysis. This work was supported in part by National Institutes of Health grants R01AI127465 and R01GM126296, the National Natural Science Foundation of China grants 31770948, 31570875 and 31200559 (S.O.) and by a research fund from the First Hospital of Jilin University. Mass spectrometry analysis was performed in the Environmental Molecular Sciences Laboratory, a US Department of Energy national scientific user facility at Pacific Northwest National Laboratory. Battelle operates the Pacific Northwest National Laboratory for the Department of Energy under contract DE-AC05-76RLO01830. The diffraction data were collected at beamline BL-17U1 of the Shanghai Synchrotron Radiation Facility.
Author information
Authors and Affiliations
Contributions
N.G. and Z.-Q.L. conceived the ideas for this work. Unless specified, N.G. and Yao Liu performed the experiments. Yao Liu, Yancheng Liu, N.G. and J.Q. performed the yeast experiments; G.M.F. and E.S.N. performed mass spectrometric analyses. X.Z., X.X., C.H., B.Z., L.Z. and S.O. determined the structures and analysed protein properties using biophysical tools. K.P. and C.D. performed HPLC analysis of nucleotide products. N.G., Yao Liu, E.S.N., S.O. and Z.-Q.L. interpreted the results. N.G., Yao Liu, S.O. and Z.-Q.L. wrote the manuscript and all authors provided editorial input.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Friedrich Förster, Elizabeth Hartland, Carsten Janke and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 Determination of the modification rate of E860 of SdeA.
a, Peak areas of the extracted-ion chromatograms (XIC) were normalized on the basis of the area of the unmodified peptide –I608IQQILANPDCIHDDHVLINGQK630–. The occupancy rate of glutamylation on the residue was calculated on the basis of the consumption of the unmodified –H855GEGTESEFSVYLPEDVALVPVK877– in samples from cells cotransfected to express GFP–SidJ compared to those of controls from cells transfected to express GFP. b, SidJ induces a 258.09-Da post-translation modification on E860 within the mART motif of SdeA. 4×Flag–mART purified from HEK293T cells coexpressing SidJ detected by silver staining (Fig. 2d) was analysed by mass spectrometric analysis. The tandem mass spectrum shows the fragmentation profile of the modified peptide –H855GEGTEGluGluSEFSVYLPEDVALVPVK877–, including ions b5 and b6, which confirms the modification site at the E860 residue. In each case, similar results were obtained in three independent experiments.
Extended Data Fig. 2 The effects of cell lysates, ATP and heat treatment of CaM on the activity of SidJ and its inhibition of the activity of all members of the SidE family.
a, Inhibition of SdeA activity does not occur in in vitro reactions containing l-glutamate or each of its two structural isomers. l-glutamate, N-acetylserine or N-methylaspartate was incubated with SdeA, SidJ and ATP for 2 h before assaying for the activity of SdeA. b, One or more factors from mammalian cells are required for SidJ to inhibit SdeA. Lysates from E. coli or HEK293T cells were added to reactions containing SdeA and SidJ for 2 h before measuring the activity of SdeA. c, Heat treatment does not completely abolish CaM activity. CaM or CaM treated by heating at 100 °C for 5 min was included in reactions that allow glutamylation of SdeA for 2 h. A cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to each reaction. Samples were resolved by SDS–PAGE and analysed for Rab33b ubiquitination after another 2 h incubation at 37 °C. d, The activity of SidJ requires ATP. His6-SdeA was incubated with GST–SidJ, l-glutamate and CaM in reactions with or without 1 mM ATP for 2 h; 4×Flag–Rab33b, NAD+ and ubiquitin were added to each reaction. After another 2-h incubation, the activity of SdeA was evaluated by the production of ubiquitinated Ra33b. Protein components in the reactions were detected by immunoblotting with specific antibodies. e, The binding of ATP by SidJ. Binding of ATP by purified SidJ was evaluated using microscale thermophoresis in which the concentration of SidJ was kept constant. Kd was determined by the NanoTemper Analysis 2.2.4 software. f, SidJ inhibits the activity of members of the SidE family. A recombinant protein of each member of the SidE family was incubated with ATP, l-glutamate and GST–SidJ in the presence or absence of CaM for 2 h, and a cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to the reactions. After an additional 2-h incubation, modification of Rab33b was detected by immunoblotting with a Flag-specific antibody. The formation of Ub-4×Flag–Rab33b is indicated by a shift in molecular mass. In each panel, data shown are one representative from at least three independent experiments with similar results.
Extended Data Fig. 3 The IQ motif of SidJ is required for its optimal response to CaM.
a, b, The IQ motif is required for the optimal activity of SidJ in response to CaM. Serially diluted CaM was preincubated with SidJ (a) or the SidJ(I841D/Q842A) mutant (b) and SdeA in the glutamylation buffer at 37 °C for 2 h. A cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to the reactions. After incubation for another 2 h at 37 °C, proteins separated by SDS–PAGE were assessed using the indicated antibodies. In each panel, data shown are one representative from at least three independent experiments with similar results. c, The SidJ(I841D/Q842A) mutant complements the intracellular growth defect of the ΔsidJ mutant. A. castellanii was infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Experiments on each strain were performed in triplicate and similar results were obtained in two independent experiments. Results are from one representative experiment performed in triplicate from three independent experiments; error bars represent s.e.m. (n = 3).
Extended Data Fig. 4 SidJ forms a stable heterodimer with CaM at a molar ratio of 1:1.
a, SidJ(∆N99) maintains the ability to inhibit SdeA activity, to a similar extent to that of full-length SidJ. SdeA was incubated with GST–SidJ or SidJ(∆N99) at indicated molar ratios in reactions containing ATP, l-glutamate, CaM for 2 h at 37 °C. A cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to each reaction for an additional 2 h at 37 °C, and the proteins resolved by SDS–PAGE were analysed with the indicated antibodies. SdeA activity was measured by the production of ubiquitinated Rab33b as indicated by a shift in molecular mass. b, Size-exclusion chromatography profiles of SidJ–CaM. Left, purified proteins were separated by a Superdex 200 Increase 10/300 column (GE Healthcare) on an AKTA pure system. Right, fractions with strong absorbance at an optical density of 260 nm (OD260) were collected and analysed by SDS–PAGE followed by detection with Coomassie brilliant blue staining. c, The heterodimer formed between SidJ(∆N99) with CaM is a monomer. Analytical ultracentrifugation analysis yielded a sedimentation coefficient of 5.770 S, and a molecular mass of approximately 96.12 kDa, which is indicative of the heterodimer of SidJ(∆N99) and CaM. In each panel, data shown are one representative from at least three independent experiments with similar results.
Extended Data Fig. 5 Overall structure of the SidJ–CaM complex in one asymmetric unit and the comparison of complex structures with or without AMP.
a, Two views of the structure of the SidJ–CaM heterodimer in the asymmetric unit displayed as a ribbon diagram (top) and with surface rendering (bottom); one of the SidJ–CaM heterodimers is coloured as shown in Fig. 4 and the other one is coloured in grey. b, Superimposition of the structures of SidJ–CaM and SidJ–CaM–AMP. The SidJ–CaM–AMP ternary complex is coloured as shown in Fig. 4d and the SidJ–CaM binary complex is coloured in grey.
Extended Data Fig. 6 Interactions between CaM and Ca2+ from the crystal structures and the role of Ca2+ in the activation of SidJ by CaM.
a, Key residues of CaM involved in the interaction with Ca2+. Ca2+ is coordinated by D21, D23, D25 and T27 of CaM, which are shown as red sticks. Ca2+ is shown as a pink sphere. Electron density of a simulated annealing Fo − Fc omit map for Ca2+ contoured at 3.0σ. b, Dialysis against 20 mM EGTA does not abolish the activity of SidJ. All proteins used in the reactions were dialysed against a buffer containing 20 mM EGTA for 14 h. SdeA was incubated with SidJ in reactions containing ATP and EGTA-dialysed CaM for 2 h at 37 °C. Reactions without SidJ were established as a control. A cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to each reaction. After further incubation for 2 h at 37 °C, proteins resolved by SDS–PAGE were analysed with the indicated antibodies. SdeA activity was measured by the production of ubiquitinated Rab33b as indicated by a shift in molecular mass. c, The activity of SidJ is not sensitive to 10 mM EGTA. SdeA was first incubated with SidJ for glutamylation with the indicated amounts of EGTA for 2 h at 37 °C. NAD+, 4×Flag–Rab33b and ubiquitin were then supplemented to the reactions, which were allowed to proceed for 2 h at 37 °C before resolution by SDS–PAGE. Rab33b modification was detected as described in b. Proteins in the reactions were detected by immunoblotting with specific antibodies. In b, c, similar results were obtained in at least three independent experiments.
Extended Data Fig. 7 The mechanism of SidJ-induced CaM-dependent self-AMPlyation and SdeA glutamylation.
a, SidJ induces self-AMPylation in a CaM-dependent manner. SidJ was incubated with 32P-α-ATP and Mg2+, with or without CaM for 2 h at 37 °C. After separation by SDS–PAGE, the incorporation of 32P-α-ATP was detected by autoradiography. b, SdeA glutamylation by SidJ interferes with SidJ self-AMPylation. SidJ was incubated with 32P-α-ATP, Mg2+ and CaM for 2 h at 37 °C. l-glutamate, SdeA and SdeA(E860D) were supplemented as stated. After separation by SDS–PAGE, the incorporation of 32P-α-ATP was detected by autoradiography. c, SdeA glutamylation by SidJ accelerates ATP hydrolysis and the release of AMP. SidJ was incubated with the indicated components for 2 h at 37 °C. Samples were analysed by HPLC. AMP and ATP were used as standards. In a–c, data shown are one representative from at least three independent experiments with similar results. d, Schematic model of SidJ-induced glutamylation and AMPylation. SidJ induces glutamylation on SdeA(E860D) when ATP and l-glutamate are supplemented into the reaction. In reactions in which l-glutamate or modifiable SdeA are not present, SidJ undergoes self-AMPylation.
Extended Data Fig. 8 Intracellular growth phenotypes associated with the ∆sidJ mutant expressing SdeA and its mutants.
a, Intracellular defects of the L. pneumophila ∆sidJ mutant can be complemented by SidJ expressed from a multicopy plasmid. The indicated strains were used to infect A. castellanii at an MOI of 0.05 and the growth of the bacteria was evaluated at 24-h intervals. Fold growth was calculated on the basis of total bacterial counts at the indicated time points and those of the 2-h time point. b, Overexpression of a SdeA mutant defective in substrate recognition inhibits intracellular growth of the ∆sidJ mutant. Intracellular growth of the indicated L. pneumophila strains in A. castellanii was evaluated as described in a. In each panel, the expression of SidJ, SdeA and its mutants in bacterial cells and their translocation into infected cells was determined by immunoblotting from total bacterial cell lysates and the saponin-soluble fraction of infected cells, with isocitrate dehydrogenase and tubulin as loading controls, respectively (right). In each case, results are from one representative experiment performed in triplicate from three independent experiments; error bars represent s.e.m. (n = 3).
Extended Data Fig. 9 SidJ functions to regulate the activity of SdeA during L. pneumophila infection.
a, SdeA(E860D) is resistant to glutamylation catalysed by SidJ. SdeA, SdeA(E860A) or SdeA(E860D) was added to reactions containing GST–SidJ, 14C-glutamate ATP and CaM and the reactions were allowed to proceed for 2 h at 37 °C. After separation by SDS–PAGE, the incorporation of 14C-glutamate was detected by autoradiography. b, Yeast toxicity induced by SdeA(E860D) cannot be suppressed by SidJ. A plasmid that directs the expression of SidJ was introduced into yeast strains expressing SdeA or SdeA(E860D) from a galactose inducible promoter, serially diluted yeast cells were spotted onto glucose or galactose medium for 2 days and the growth of the cells was evaluated by imaging (top). The expression of SidJ, SdeA and SdeA(E860D) was determined by immunoblotting with specific antibodies. The PGK1 (3-phosphoglyceric phosphokinase-1) was analysed as a loading control (bottom). c, SdeA(E860D) still ubiquitinates Rab33b. Reactions containing the indicated components were allowed to proceed for 2 h at 37 °C, samples were then resolved by SDS–PAGE and ubiquitination of Rab33b was assessed by immunoblotting with a Flag-specific antibody to detect the production of modified Rab33b with a higher molecular mass. d, SdeA(E860D)-mediated protein ubiquitination in mammalian cells is insensitive to SidJ. HEK293T cells were transfected to express the indicated proteins for 16–18 h. Cleared cell lysates were subjected to SDS–PAGE and immunoblotting with an HA-specific antibody to detect proteins ubiquitinated by 3×HA–Ub-AA. The amounts of SdeA, SdeA(E860D) and SidJ were assessed by antibodies specific for these proteins. Note that coexpression of SidJ reduced the ubiquitination induced by SdeA but not by SdeA(E860D). In a–d, data shown are one representative from at least three independent experiments with similar results. e, The effects of SidJ on intracellular growth defect caused by overexpression of SdeA or SdeA(E860D). The indicated L. pneumophila strains were used to infect A. castellanii at an MOI of 0.05 and the growth of the bacteria was evaluated at 24-h intervals. Fold growth was calculated on the basis of total bacterial counts at the indicated time points. Note the difference between strain ∆sidJ (pSdeA) and ∆sidJ (pSdeA, pSidJ). The growth defect caused by overexpressing the SdeA(E860D) mutant cannot be rescued by SidJ. The amounts of relevant proteins in bacterial cells and in infected cells were analysed by immunoblotting from total bacterial cell lysates and the saponin-soluble fraction of infected cells, with isocitrate dehydrogenase and tubulin as loading controls, respectively (right). Results showen are from one representative experiment performed in triplicate from three independent experiments; error bars represent s.e.m. (n = 3).
Supplementary information
Supplementary Information
This file contains uncropped blots used in main figures and extended data figures.
Rights and permissions
About this article
Cite this article
Gan, N., Zhen, X., Liu, Y. et al. Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase. Nature 572, 387–391 (2019). https://doi.org/10.1038/s41586-019-1439-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-019-1439-1
This article is cited by
-
Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain
Nature Communications (2024)
-
An expanded lexicon for the ubiquitin code
Nature Reviews Molecular Cell Biology (2023)
-
Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila
Nature Communications (2022)
-
A secreted effector with a dual role as a toxin and as a transcriptional factor
Nature Communications (2022)
-
Polyglutamylation: biology and analysis
Amino Acids (2022)
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.
