Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1,2,3,4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6,7,8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hershko, A., Ciechanover, A. & Varshavsky, A. The ubiquitin system. Nat. Med. 6, 1073–1081 (2000).
Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).
Hurley, J. H. & Stenmark, H. Molecular mechanisms of ubiquitin-dependent membrane traffic. Annu. Rev. Biophys. 40, 119–142 (2011).
Haglund, K. & Dikic, I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J. Cell Sci. 125, 265–275 (2012).
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).
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.e1613 (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).
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).
Zhou, Y. & Zhu, Y. Diversity of bacterial manipulation of the host ubiquitin pathways. Cell. Microbiol. 17, 26–34 (2015).
Lin, Y. H. & Machner, M. P. Exploitation of the host cell ubiquitin machinery by microbial effector proteins. J. Cell Sci. 130, 1985–1996 (2017).
Qiu, J. & Luo, Z. Q. Legionella and Coxiella effectors: strength in diversity and activity. Nat. Rev. Microbiol. 15, 591–605 (2017).
Hsu, F. et al. The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling. Proc. Natl Acad. Sci. USA 111, 10538–10543 (2014).
Luo, X. et al. Structure of the Legionella virulence factor, SidC reveals a unique PI(4)P-specific binding domain essential for its targeting to the bacterial phagosome. PLoS Pathog. 11, e1004965 (2015).
Price, C. T. et al. Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa. PLoS Pathog. 5, e1000704 (2009).
Kubori, T., Hyakutake, A. & Nagai, H. Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol. Microbiol. 67, 1307–1319 (2008).
Kubori, T., Shinzawa, N., Kanuka, H. & Nagai, H. Legionella metaeffector exploits host proteasome to temporally regulate cognate effector. PLoS Pathog. 6, e1001216 (2010).
Ensminger, A. W. & Isberg, R. R. E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates. Infect. Immun. 78, 3905–3919 (2010).
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).
Jeong, B. R. et al. Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity. J. Biol. Chem. 286, 43272–43281 (2011).
Tsurumura, T. et al. Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex. Proc. Natl Acad. Sci. USA 110, 4267–4272 (2013).
Simon, N. C., Aktories, K. & Barbieri, J. T. Novel bacterial ADP-ribosylating toxins: structure and function. Nat. Rev. Microbiol. 12, 599–611 (2014).
Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).
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).
Rinaldo, S. et al. Structural basis of functional diversification of the HD-GYP domain revealed by the Pseudomonas aeruginosa PA4781 protein, which displays an unselective bimetallic binding site. J. Bacteriol. 197, 1525–1535 (2015).
Kalayil, S. et al. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination. Nature https://doi.org/10.1038/s41586-018-0145-8 (2018).
Klumpp, S. & Krieglstein, J. Phosphorylation and dephosphorylation of histidine residues in proteins. Eur. J. Biochem. 269, 1067–1071 (2002).
Stock, A. M., Robinson, V. L. & Goudreau, P. N. Two-component signal transduction. Annu. Rev. Biochem. 69, 183–215 (2000).
Burstein, D. et al. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires. Nat. Genet. 48, 167–175 (2016).
Raasi, S. & Pickart, C. M. Ubiquitin chain synthesis. Methods Mol. Biol. 301, 47–55 (2005).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).
Trapani, S. & Navaza, J. AMoRe: classical and modern. Acta Crystallogr. D 64, 11–16 (2008).
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Johnson, B. A. & Blevins, R. A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).
Smolsky, I. L. et al. Biological small-angle X-ray scattering facility at the Stanford synchrotron radiation laboratory. J. Appl. Crystallogr. 40, s453–s458 (2007).
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).
Schneidman-Duhovny, D., Hammel, M. & Sali, A. FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 38, W540–W544 (2010).
Weinkam, P., Pons, J. & Sali, A. Structure-based model of allostery predicts coupling between distant sites. Proc. Natl Acad. Sci. USA 109, 4875–4880 (2012).
Martí-Renom, M. A. et al. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29, 291–325 (2000).
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
Daniels, C. M., Ong, S. E. & Leung, A. K. Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J. Proteome Res. 13, 3510–3522 (2014).
We acknowledge L. Pollack’s group for SAXS data collection. This work is supported by National Institute of Health (NIH) grants 5R01GM116964 (Y.M.), R01AI127465 (Z.-Q.L.), R01GM088055 (R.E.K.), 1R01GM098503-05 (P.S.B.), and 1 F32 GM120797 (K.H.R.). The X-ray data were collected at Cornell High Energy Synchrotron Source. CHESS is supported by the NSF and NIH/National Institute of General Medical Sciences (NIGMS) via NSF award DMR-1332208, and the MacCHESS resource is supported by NIH/NIGMS award GM-103485. Some SAXS data were collected at Stanford Synchrotron Radiation Lightsource. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (including P41GM103393).
Nature thanks K. Gehring and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Phosphoribosyl-linked ubiquitination catalysed by SdeA involves two enzymatic activities of SdeA. First, using its mART activity, SdeA catalyses the ADP-ribosylation of Ub to generate ADPR-Ub by consuming an NAD+ molecule. Second, SdeA catalyses the conjugation of ADPR-Ub to a serine residue of substrate proteins via its PDE activity to generate protein–PR-Ub and AMP. In the absence of substrate proteins, the PDE domain of SdeA can simply hydrolyse ADPR-Ub to PR-Ub and AMP using a water molecule.
a, Model of the PDE domain of SdeA in ribbon representation. Two invariable histidine residues (H277 and H407) are shown in stick representation and labelled. b, Surface representation of the PDE domain. The two invariable histidine residues (shown in red) are situated at the bottom of a deep groove. c, The PDE domain from a Legionella effector (lpg1496). Notably the all α-helical structural core of the PDE domains is easy to superimpose onto that of SdeA with a root mean square deviation (r.m.s.d.) of 1.9 Å over 225 aligned Cα atoms. A prominent difference between the two PDE domains is that some loops (indicated by dashed outlines) connecting the α-helices vary both in primary sequence and in length (Extended Data Fig. 3). d, Surface residue conservation analysis of the PDE domain. The conservation is calculated using the ConSurf server with the most conserved residues coloured in purple and the least conserved residues in cyan. Note that the catalytic groove is enriched with the most conserved residues.
Extended Data Fig. 3 Multiple sequence alignment of selected PDE domains from the SidE family effectors.
Representative sequences corresponding to the PDE domain of SdeA (amino acids 222–502) were aligned using the MultAlin online server (http://www.bioinformatics.org/sms/index.html). Secondary structural elements are drawn above the alignment. The numbering for the SdeA sequence is marked on the top of the alignment and the numbering for the SdeD sequence is marked below. Variable loop regions are outlined with dashed squares. Conserved residues located within the catalytic groove are highlighted with purple dots. In particular, three essential catalytic residues (H277, H407 and E340) are highlighted with red stars below the sequences. SdeD residues that are in close contact with Ub1 (Fig. 3a) are marked by blue triangles at the bottom of the sequences and the predicted Ub1-interacting residues of the PDE domain of SdeA (Fig. 3e) are depicted by red triangles on the top of the sequences. Amongst the potential Ub1-interacting residues, V414, E454 and E465 of SdeA used in mutagenesis studies in Fig. 3f, g are marked with solid red triangles. Entrez database accession numbers are as follows: SdeA, GI: 1064303039; SidE, GI: 52840489; SdeB, GI: 52842367; SdeC, GI: 52842370; lpg2154, GI: 52842368; and SdeD, GI: 52842717.
Extended Data Fig. 4 Structural comparison of the SdeA mART domain with other mART domains from bacterial toxins.
a, Model of the main lobe of the SdeA mART domain in ribbon representation. The main lobe is composed of two nearly perpendicular β-sheets forming a two-layered β-sandwich core. Residues comprising the three mART catalytic signature motifs: (F/Y)-(R/H), STS and EXE motif are shown in sticks. b. HopU1 from P. syringae (PDB ID: 3U0J) in ribbon representation. c, Structural superimposition of the mART domains from SdeA (gold) and HopU1 (blue). d, Iota-toxin from C. perfringens (PDB ID: 4H03). e, Iota-toxin in complex with NAD+ (red spheres). f, Structural overlay of the mART domains from SdeA (gold) and iota-toxin (cyan). g, A cartoon diagram of the α-helical lobe of the SdeA mART domain. The α-helical lobe consists of eight α-helices. Three structurally conserved α-helices (α6–8) are coloured in brown. h, A cartoon diagram of the α-helical lobe of HopU1, the three equivalent α-helices (α4–6) are highlighted in blue. i, Structural overlay of the α-helical lobe of SdeA and HopU1.
Representative sequences corresponding to the mART domains of SdeA (amino acids 593–904) were aligned using MultAlin. Secondary structural elements (cyan for the α-helical lobe and gold for the main lobe of the mART domain) are drawn above the alignment. The numbering for the SdeA sequence is marked on the top of the alignment. Residues comprising the catalytically important (F/Y)-(R/H), STS and EXE motifs are marked with red stars. Residues in the α-helical lobe, which form—or are close to—the conserved surface patch and are essential for the mART activity (Extended Data Fig. 7), are marked with purple triangles. D622, which is conserved but has no effect on the mART activity is marked with a green triangle. Entrez database accession numbers are as follows: SdeA, GI: 1064303039; SidE, GI: 52840489; SdeB, GI: 52842367; SdeC, GI: 52842370; SidE Legionella cincinnatiensis, GI: 966421657; LLO_3095, GI: 489730495; SidE Legionella gratiana, GI: 966468332; SidE Legionella santicrucis, GI: 966496250; LLO_0424, GI: 502743808.
Extended Data Fig. 6 The α-helical lobe of SdeA mART domain has an extended conformation compared to other mART proteins.
a, Structural superimposition of SdeA onto the HopU1 structure referenced on the main lobe of the mART domain. SdeA is coloured using the same scheme as Fig. 1b. The main lobe of HopU1 is coloured in blue and its α-helical lobe is in grey. The α-helical lobe of the SdeA mART is extended away from the main lobe whereas its counterpart in HopU1 packs in close contact with the main lobe. b, Structural model of SdeA with the α-helical lobe in a closed conformation. The positioning of the α-helical lobe was based on a structural overlay of the three structurally conserved α helices identified in all mART domains (Extended Data Fig. 4g–i). c, Experimental and theoretical SAXS curves for SdeA-core and the resulting best-fit AllosMod structure for the determined structure (open) and modelled closed conformation, with residual plots shown below. Best fit χ2 values are indicated. d, Overlay of the determined SdeA-core structure (PDE, green; mART main lobe and α-helical lobe, yellow) and best-fit AllosMod structures for the open (magenta) and closed (cyan) conformations. e, Summary of the experimentally derived SAXS parameters for SdeA-core, AllosMod derived best-fit Rg and average FOXS χ2 for the five best-fitting AllosMod models compared to the experimental SAXS curve. The program Primus was used to calculate the radius of gyration (Rg) and maximum linear dimension (Dmax). Kratky plot (I(q)q2 versus q), and distance-distribution plot P(r) obtained from GNOM are shown. f, Overlay of SdeA-core SAXS curves in the presence of 4.7 mM NAD+ (10× protein concentration), with corresponding Guinier Rg values. Data shown in c, e and f are representative of two biologically independent experiments.
Extended Data Fig. 7 The α-helical lobe of SdeA mART domain is indispensable for Ub ADP-ribosylation.
a, Surface representation of residue conservation of SdeA (the most conserved residues are shown in purple and the least conserved residues in cyan). Surface residue conservation was calculated using the ConSurf server. An expanded view of a surface cluster that consists of the most conserved residues on the α-helical lobe is shown on the right. b, Analysis of in vitro ubiquitin-modification assays by SdeA mutants carrying mutations on the α-helical lobe. The reaction products were analysed using native PAGE with Coomassie blue stain (top) and SDS–PAGE with Pro-Q phosphoprotein stain (bottom). c, SDS–PAGE analysis of the proteins in the reaction mixture. Data shown in b and c are representative of three independent experiments. Uncropped gels are shown in Supplementary Fig. 1.
a, NMR 1H–15N HSQC TROSY spectral overlay of 150 μM Ub (black) in the presence or absence of 300 μM SdeA PDE domain (cyan). Ub binds very weakly to SdeA as manifested by minimal changes in 15NH peaks of Ub. b. Spectral overlay of 150 μM Ub (black) with 75 μM SdeD PDE. Ub binds with higher affinity to SdeD as evidenced by peak broadening and/or disappearance of Ub resonances. c, Residues whose resonances are most affected by the presence of SdeD are mapped in red on a cartoon structure of Ub. d, PDE domain of SdeD (grey) shown in ribbon representation. Two invariable histidine residues (H67 and H189) are shown in stick representation (cyan). The variable loop unique to SdeD is outlined. e, Structural overlay of the PDE domain of SdeD (grey) and the PDE domain of SdeA (green). The overall structures of these two PDE domains are very similar with an r.m.s.d. of 1.73 Å over 251 overlaid Cα atoms. f, Two orthogonal views of the SdeD PDE domain in complex with two Ub molecules in ribbon representation: Ub1 (cyan) and Ub2 (blue). Ub1 binds at the opening of the PDE catalytic groove with its R42 side chain sticking into the groove. Ub2 binds a region on the opposite side of the catalytic groove. g, Structural superimposition of SdeA onto the SdeD PDE–Ub complex referenced on the PDE domain. The PDE domain of SdeA is shown in green and the mART domain is shown in gold. Note that Ub1 shows no conflicting contacts against the superimposed SdeA molecule whereas the Ub2 binding site largely overlaps with the space occupied by the mART domain in SdeA. This analysis suggests that the binding of the PDE domain of SdeD to Ub1 is probably applicable to the PDE domain of SdeA; however, the second Ub-binding site observed in SdeD might not exist in SdeA. Experiments in a and b were repeated independently two times.
a, SdeD PDE domain H67A mutant in complex with both ADPR-Ub and unmodified Ub. The crystal was obtained by mixing the SdeD PDE H67A mutant, ADPR-Ub, and Ub in a 1:2:3 molar ratio (see the ‘Protein crystallization’ section of the Methods for details). The PDE domain is shown in grey, the bound ADPR-Ub is shown in cyan and the unmodified Ub is shown in blue. The unmodified Ub binds a region identical to Ub2 found in the SdeD–Ub complex shown in Extended Data Fig. 7d. ADPR-Ub binds in a mode that is similar to that of Ub1 in the SdeD–Ub complex with the ADPR moiety fitting into the catalytic groove. b, An orthogonal view of a. c, d, Two orthogonal views of the complex shown in a in surface representation. Note that the ADPR-moiety shown in light green fits deeply into the catalytic groove. e, The density was generated by refinement against the structural model without the ADPR portion. The Fo − Fc difference map is shown in green and contoured at 1σ.
About this article
Cite this article
Akturk, A., Wasilko, D.J., Wu, X. et al. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector. Nature 557, 729–733 (2018). https://doi.org/10.1038/s41586-018-0147-6
In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
Frontiers in Cellular and Infection Microbiology (2020)
Insights into catalysis and regulation of non-canonical ubiquitination and deubiquitination by bacterial deamidase effectors
Nature Communications (2020)
Molecular Cell (2020)
Nature Chemical Biology (2020)