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• W2983004433 abstract "•Phosphoribosyl serine ubiquitination can be reverted by DupA and DupB•DUP specifically binds to and cleaves PR-ubiquitin from serine on substrates•Catalytically inactive DupA mutants can capture PR-ubiquitinated proteins•PR ubiquitination on multiple ER structural proteins causes ER fragmentation The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection. The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection. Ubiquitination is one of the most versatile post-translational modifications, controlling a wide variety of cellular processes (Hochstrasser, 2009Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (575) Google Scholar). In most cases, the carboxy terminus of ubiquitin (Ub) is covalently linked to the ε-amino (a primary amine) group of one or more lysines on substrates. Subsequent additions of further Ub moieties create polymers of Ub, which have diverse structures and functions (Yau and Rape, 2016Yau R. Rape M. The increasing complexity of the ubiquitin code.Nat. Cell Biol. 2016; 18: 579-586Crossref PubMed Scopus (529) Google Scholar). These Ub structures can be recognized by specific receptors that contain Ub-binding domains (UBDs) that can result in the delivery of ubiquitinated substrate to the proteasome for degradation or to selective autophagy pathways to changes in protein function and/or cellular localization (Dikic, 2017Dikic I. Proteasomal and Autophagic Degradation Systems.Annu. Rev. Biochem. 2017; 86: 193-224Crossref PubMed Scopus (500) Google Scholar). The mechanism underlying the ubiquitination process is well established. It involves a cascade of three enzymes: E1-Ub activating enzyme, E2-Ub conjugating enzyme, and E3-Ub ligase. Specific enzymes called deubiquitinases (DUBs) cleave off Ub from substrates and regulate the abundance of ubiquitinated proteins (Clague et al., 2019Clague M.J. Urbé S. Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function.Nat. Rev. Mol. Cell Biol. 2019; 20: 338-352Crossref PubMed Scopus (268) Google Scholar). Given the importance of Ub signaling, a considerable number of pathogens utilize virulence factors that modulate Ub and autophagy systems to promote pathogenicity (Grohmann et al., 2018Grohmann E. Christie P.J. Waksman G. Backert S. Type IV secretion in Gram-negative and Gram-positive bacteria.Mol. Microbiol. 2018; 107: 455-471Crossref PubMed Scopus (165) Google Scholar, Hicks and Galán, 2013Hicks S.W. Galán J.E. Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors.Nat. Rev. Microbiol. 2013; 11: 316-326Crossref PubMed Scopus (88) Google Scholar, Llosa et al., 2009Llosa M. Roy C. Dehio C. Bacterial type IV secretion systems in human disease.Mol. Microbiol. 2009; 73: 141-151Crossref PubMed Scopus (53) Google Scholar, Maculins et al., 2016Maculins T. Fiskin E. Bhogaraju S. Dikic I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion.Cell Res. 2016; 26: 499-510Crossref PubMed Scopus (68) Google Scholar, Qiu and Luo, 2017Qiu J. Luo Z.-Q. Legionella and Coxiella effectors: strength in diversity and activity.Nat. Rev. Microbiol. 2017; 15: 591-605Crossref PubMed Scopus (137) Google Scholar). This is clearly demonstrated by Legionella pneumophila, a Gram-negative bacterium that causes Legionnaires’ disease and possesses the largest number of documented bacterial effectors among intracellular bacterial pathogens (Burstein et al., 2016Burstein D. Amaro F. Zusman T. Lifshitz Z. Cohen O. Gilbert J.A. Pupko T. Shuman H.A. Segal G. Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires.Nat. Genet. 2016; 48: 167-175Crossref PubMed Scopus (152) Google Scholar). For example, the Legionella effectors LegU1 and LeuAU13 serve as F-box-containing E3 ligases that interact with host Cul1-Skp1 and ubiquitinate BAT3, a host chaperone protein (Ensminger and Isberg, 2010Ensminger A.W. Isberg R.R. E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates.Infect. Immun. 2010; 78: 3905-3919Crossref PubMed Scopus (93) Google Scholar). Another effector is LubX, a RING and U-box type E3 ligase, which, in conjunction with the host E2 enzymes UbcH5a or UbcH5c, ubiquitinates host Clk1 kinase (Kubori et al., 2008Kubori T. Hyakutake A. Nagai H. Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions.Mol. Microbiol. 2008; 67: 1307-1319Crossref PubMed Scopus (173) Google Scholar, Quaile et al., 2015Quaile A.T. Urbanus M.L. Stogios P.J. Nocek B. Skarina T. Ensminger A.W. Savchenko A. Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila.Structure. 2015; 23: 1459-1469Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). More recently, Legionella pneumophila was also shown to utilize a non-canonical type of ubiquitination through the action of enzymes belonging to the SidE family of effectors (SdeA, SdeB, SdeC, and SidE) (Bhogaraju et al., 2016Bhogaraju S. Kalayil S. Liu Y. Bonn F. Colby T. Matic I. Dikic I. Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.Cell. 2016; 167: 1636-1649.e13Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Qiu et al., 2016Qiu J. Sheedlo M.J. Yu K. Tan Y. Nakayasu E.S. Das C. Liu X. Luo Z.-Q. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.Nature. 2016; 533: 120-124Crossref PubMed Scopus (197) Google Scholar). This NAD-dependent modification involves the conjugation of Ub via a phosphoribosyl (PR) moiety to serine residues of host substrates (Bhogaraju et al., 2016Bhogaraju S. Kalayil S. Liu Y. Bonn F. Colby T. Matic I. Dikic I. Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.Cell. 2016; 167: 1636-1649.e13Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Qiu et al., 2016Qiu J. Sheedlo M.J. Yu K. Tan Y. Nakayasu E.S. Das C. Liu X. Luo Z.-Q. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.Nature. 2016; 533: 120-124Crossref PubMed Scopus (197) Google Scholar). SidE-type enzymes contain two intrinsic enzymatic domains: the mono ADP-ribosyl transferase (mART) domain that utilizes NAD to transfer ADP-ribose (ADPR) on Arg42 of Ub and the phosphodiesterase (PDE) domain that cleaves ADPR-Ub to PR-Ub and conjugates PR-Ub to substrate serines (Akturk et al., 2018Akturk A. Wasilko D.J. Wu X. Liu Y. Zhang Y. Qiu J. Luo Z.-Q. Reiter K.H. Brzovic P.S. Klevit R.E. Mao Y. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.Nature. 2018; 557: 729-733Crossref PubMed Scopus (35) Google Scholar, Dong et al., 2018Dong Y. Mu Y. Xie Y. Zhang Y. Han Y. Zhou Y. Wang W. Liu Z. Wu M. Wang H. et al.Structural basis of ubiquitin modification by the Legionella effector SdeA.Nature. 2018; 557: 674-678Crossref PubMed Scopus (38) Google Scholar, Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar, Wang et al., 2018Wang Y. Shi M. Feng H. Zhu Y. Liu S. Gao A. Gao P. Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE.Cell. 2018; 173: 1231-1243.e16Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Among the known PR-ubiquitinated substrates are several ER-associated Rab GTPases and reticulon 4 (Rtn4). Upon infection, Legionella pneumophila regulates dynamics of membranes to create a Legionella-containing vacuole (LCV) where they can reside and avoid the host defense system. PR ubiquitination has been shown to impair GTP-loading and GTP-hydrolysis activity of Rab GTPases (Qiu et al., 2016Qiu J. Sheedlo M.J. Yu K. Tan Y. Nakayasu E.S. Das C. Liu X. Luo Z.-Q. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors.Nature. 2016; 533: 120-124Crossref PubMed Scopus (197) Google Scholar) and tubular ER rearrangements and potential fragmentation of ER in order to promote proliferation of bacteria in the LCV (Kotewicz et al., 2017Kotewicz K.M. Ramabhadran V. Sjoblom N. Vogel J.P. Haenssler E. Zhang M. Behringer J. Scheck R.A. Isberg R.R. A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication.Cell Host Microbe. 2017; 21: 169-181Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Recent evidence also shows a role of SidE family effectors in regulating mTORC1 activity through PR ubiquitination of Rag GTPases on the lysosome (De Leon et al., 2017De Leon J.A. Qiu J. Nicolai C.J. Counihan J.L. Barry K.C. Xu L. Lawrence R.E. Castellano B.M. Zoncu R. Nomura D.K. et al.Positive and Negative Regulation of the Master Metabolic Regulator mTORC1 by Two Families of Legionella pneumophila Effectors.Cell Rep. 2017; 21: 2031-2038Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Moreover, the Legionella effector SidJ has been proposed to act as a deubiquitinase for both conventional and PR-linked ubiquitination (Qiu et al., 2017Qiu J. Yu K. Fei X. Liu Y. Nakayasu E.S. Piehowski P.D. Shaw J.B. Puvar K. Das C. Liu X. Luo Z.Q. A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination.Cell Res. 2017; 27: 865-881Crossref PubMed Scopus (48) Google Scholar); however, recent findings indicate that SidJ acts as a glutamylase that inhibits SidE enzymes by targeting the catalytic site of the ART domains (Bhogaraju et al., 2019Bhogaraju S. Bonn F. Mukherjee R. Adams M. Pfleiderer M.M. Galej W.P. Matkovic V. Lopez-Mosqueda J. Kalayil S. Shin D. Dikic I. Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation.Nature. 2019; 572: 382-386Crossref PubMed Scopus (54) Google Scholar, Black et al., 2019Black M.H. Osinski A. Gradowski M. Servage K.A. Pawłowski K. Tomchick D.R. Tagliabracci V.S. Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases.Science. 2019; 364: 787-792Crossref PubMed Scopus (61) Google Scholar, Gan et al., 2019Gan N. Zhen X. Liu Y. Xu X. He C. Qiu J. Liu Y. Fujimoto G.M. Nakayasu E.S. Zhou B. et al.Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.Nature. 2019; 572: 387-391Crossref PubMed Scopus (53) Google Scholar). Despite these findings, critical questions related to the spectrum of PR-ubiquitinated substrates and the associated functional consequences as well as the dynamics of PR ubiquitination remain to be explored. In this study, we address these issues by identifying two bacterial effectors encoding deubiquitinases for PR-linked ubiquitination (DUPs), which counteract the activity of SidE ligases by removing PR-ubiquitin from substrate serines. We also provide biophysical and structural explanations for their specificity toward PR-ubiquitinated substrates. Moreover, based on their strong binding affinity to PR-ubiquitinated substrates, we have engineered an inactive DupA variant that acts as a trapping mutant for endogenously PR-ubiquitinated substrates in Legionella-infected cells. This approach enabled us to identify multiple classes of PR-ubiquitinated substrates. We also show that PR ubiquitination is required for ER fragmentation and ER recruitment to LCV upon Legionella infection. Collectively, these findings provide invaluable insights into Legionella-mediated PR ubiquitination of host proteins and shed light on the functional relevance of this modification upon infection. The transfer of PR-Ub to substrate serine residues by SidEs is mediated by their PDE domains (Akturk et al., 2018Akturk A. Wasilko D.J. Wu X. Liu Y. Zhang Y. Qiu J. Luo Z.-Q. Reiter K.H. Brzovic P.S. Klevit R.E. Mao Y. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.Nature. 2018; 557: 729-733Crossref PubMed Scopus (35) Google Scholar, Bhogaraju et al., 2016Bhogaraju S. Kalayil S. Liu Y. Bonn F. Colby T. Matic I. Dikic I. Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.Cell. 2016; 167: 1636-1649.e13Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar), which resemble classical HD (histidine and aspartate) domains (Aravind and Koonin, 1998Aravind L. Koonin E.V. The HD domain defines a new superfamily of metal-dependent phosphohydrolases.Trends Biochem. Sci. 1998; 23: 469-472Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, Morar et al., 2015Morar M. Evdokimova E. Chang C. Ensminger A.W. Savchenko A. Crystal structure of the Legionella pneumophila Lem10 effector reveals a new member of the HD protein superfamily.Proteins. 2015; 83: 2319-2325Crossref PubMed Scopus (3) Google Scholar). Based on sequence similarity, we identified four additional SidE-like PDE-containing Legionella proteins (Lpg1496, Lpg2523, Lpg2154 (or LaiE), and Lpg2509 (LaiF or SdeD); Figures 1A and S8). Sequence alignment revealed that the catalytic residues of the SdeA PDE domain (E340, H277, and H407) (Akturk et al., 2018Akturk A. Wasilko D.J. Wu X. Liu Y. Zhang Y. Qiu J. Luo Z.-Q. Reiter K.H. Brzovic P.S. Klevit R.E. Mao Y. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.Nature. 2018; 557: 729-733Crossref PubMed Scopus (35) Google Scholar, Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar) are highly conserved in all eight PDE-containing Legionella proteins. Despite this high conservation, incubation of ADPR-Ub with the newly identified PDE-containing proteins did not result in autoubiquitination and/or Rab33b ubiquitination (Figure 1B, left). Instead, LaiE and LaiF (Luo and Isberg, 2004Luo Z.-Q. Isberg R.R. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer.Proc. Natl. Acad. Sci. USA. 2004; 101: 841-846Crossref PubMed Scopus (357) Google Scholar) PDE domains processed the ADPR-Ub but did not transfer the PR-Ub to the substrate in vitro (Figure 1B, left). Importantly, these PDE domains cleaved PR-ubiquitinated Rab33b (Rab33b-PR-Ub) in vitro (Figure 1B, right) and multiple PR-ubiquitinated substrates in cells (Figure 1C). As such, we renamed these Legionella effectors as DUPs: DupA/LaiE and DupB/LaiF. Moreover, both DupA and DupB specifically cleaved PR-ubiquitinated substrates but not canonical lysine-linked ubiquitination substrates (Figure 1C). Further biochemical analyses also revealed that the released Ub species were stained by phosphoprotein staining solution (Figure 1D), indicating that DupA and DupB cleaved the bond between PR-Ub and the substrate serine residue. This finding was further confirmed by mass spectrometry analysis (Figures S1A and 1E). To examine whether there are other proteins cleaving PR-Ub from serine, we generated a Legionella strain without DUPs and mixed lysates with PR-ubiquitinated Rab33b. Depletion of both DUPs, but not SidJ, which has been previously suggested to serve as a PR-ubiquitin specific deubiquitinase, failed to hydrolyze PR-ubiquitin from Rab33b (Figure 1F). Collectively, our data establish a new class of deubiquitinases specific for PR ubiquitination that includes DupA and DupB. To elucidate the molecular mechanism of DUPs, we determined the crystal structure of DupA4-345 (PDB: 6RYB, Figure 2A). The overall structure of DupA resembled the PDE domains of the SidE family ligases SdeA (Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar) and SidE (Wang et al., 2018Wang Y. Shi M. Feng H. Zhu Y. Liu S. Gao A. Gao P. Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE.Cell. 2018; 173: 1231-1243.e16Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) as well as the PDE domain of DupB (Akturk et al., 2018Akturk A. Wasilko D.J. Wu X. Liu Y. Zhang Y. Qiu J. Luo Z.-Q. Reiter K.H. Brzovic P.S. Klevit R.E. Mao Y. Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.Nature. 2018; 557: 729-733Crossref PubMed Scopus (35) Google Scholar). The three catalytic residues from SdeA and DupB PDE domains were also highly conserved in the core of DupA (H67, E126, and H189), while several loop regions differed (Figure 2A). Varying the length of the corresponding SdeA loop, deleting the loop regions from both SdeA and DupA PDE domains, or swapping the loop region did not affect the function of either PDE domains (Figures S1B–S1D). In contrast, mutation of the three conserved catalytic residues from both DupA and DupB resulted in impaired cleavage activity (Figures 2B–2D, S1E, and S1F). Moreover, after 5 min of reaction, we detected a labile and heat-sensitive His-Ub intermediate on the DupA His-189-Asn mutant, while wild-type (WT) DupA displayed heat-stable auto-PR ubiquitination , which was further cleaved by DupA at later time points (Figures 2E, 2F, and S1G). This suggests that, similar to the SdeA PDE domain, DupA utilizes a histidine-based intermediate reaction to catalyze the hydrolysis of ADPR-Ub (Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar). Thus, the PDE domains of SidEs and DupA/B may share the same catalytic residues to mediate opposite reactions: PR-Ub transfer to substrates and removal of PR-Ub from substrates (deubiquitination), respectively. We next monitored both catalytic reactions over an extended time (Figures 2F and S1G). Upon incubation of ADPR-Ub and Rab33b substrate with DupA, small amounts of DupA autoubiquitination and Rab33b PR ubiquitination were detected at the very beginning of the reaction (5–15 min), which declined at later time points (30–60 min). This indicates that the DupA PDE domain, a strong deubiquitinase (or hydrolase), also has weak transferase activity in vitro involving the hydrolysis of ADPR-Ub and the transfer of PR-Ub to a substrate. To reveal the atomic basis underlying the bias of DupA toward deubiquitinase activity, we sought to determine the structure of enzymatically inactive DupA (H67A) in complex with a PR-ubiquitinated substrate. A Rtn4 peptide, acting as a minimal ubiquitination substrate of SdeA (Kalayil et al., 2018Kalayil S. Bhogaraju S. Bonn F. Shin D. Liu Y. Gan N. Basquin J. Grumati P. Luo Z.-Q. Dikic I. Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.Nature. 2018; 557: 734-738Crossref PubMed Scopus (44) Google Scholar), was modified to harbor only one target serine residue (Figures S2A and S2B). Crystals of DupA H67A and PR-ubiquitinated Rtn4 peptide diffracted up to 2.0 Å, and molecular replacement revealed the densities for both Ub and DupA but not for the Rtn4 peptide, likely due to the flexibility of the peptide (PDB: 6RYA, Figure S2C). Superimposition of Ub in our DupA-Ub structure with the available structures of Ub complexed with the SidE PDE domain or DupB (PDB: 5ZQ3 and 6B7O, respectively) revealed a different orientation of Ub toward the conserved catalytic pocket (Figures 3A and S2D). In particular, DupA/B-Ub structures displayed a closed conformation with Ub due to extensive electrostatic interactions, while the SidE PDE domain lacked the corresponding residues (Figure 3B). Additionally, DupA H67A effectively interacted with Ub, ADPR-Ub, and PR-ubiquitinated substrates, whereas the SdeA PDE domain H277A only co-precipitated unmodified Ub (Figure 3C). Accordingly, DupA displayed a strong binding affinity and high kon to Ub, ADPR-Ub, and PR-ubiquitinated peptides, while the SdeA PDE domain showed weak affinity to Ub and ADPR-Ub (Figures 3D, 3E, and S2E; Table 1). Unmodified Ub had similar residence time (1/koff) on both DupA and SdeA PDE domains; however, ADPR-Ub exhibited reduced residence time on the SdeA PDE domain (Figure S2F; Table 1). Moreover, the SdeA PDE domain did not bind to the PR-ubiquitinated serine peptide, whereas DupA maintained a strong binding affinity. These results were also confirmed by NMR titration (Figure S3).Table 1Binding Kinetics of DupA and SdeA PDE to Ub Specieskon ± SEMaSEM, standard error of mean (102 M−1s−1)koff ± SEMaSEM, standard error of mean (10−5 s−1)Residence Time (1/koff, min)Kd ± SEMaSEM, standard error of mean (nM)R2bR2, goodness of the curve fit between experimental data and mathematical 1:1 binding curveDupAUb102 ± 0.746.59 ± 0.29253 ± 116.46 ± 0.290.99ADPR-Ub188 ± 2.193.46 ± 0.41482 ± 571.84 ± 0.220.97PR-ubiquitinated Rtn4 peptide139 ± 1.3112.2 ± 0.35137 ± 3.98.75 ± 0.270.98Rtn4 Peptide3.76 ± 0.0433.4 ± 0.3650 ± 0.5887 ± 13.90.98SdeA PDEUb5.79 ± 0.067.34 ± 0.46227 ± 14127 ± 8.140.98ADPR-Ub1.71 ± 0.0555.0 ± 0.9230 ± 0.53220 ± 1040.96PR-ubiquitinated Rtn4 peptideNDRtn4 Peptide7.89 ± 0.1058.4 ± 0.4129 ± 0.2740 ± 10.80.97a SEM, standard error of meanb R2, goodness of the curve fit between experimental data and mathematical 1:1 binding curve Open table in a new tab To provide further insights into the dynamics of this reaction, we performed MD simulation of the PR-ubiquitinated Rtn4 peptide with either the DupA or SdeA PDE domains (Figure S4; Videos S1, S2, S3, and S4). We initiated the simulation by locating Ub outside the catalytic pocket via superimposing the PR-ubiquitinated peptide with the SidE-Ub complex structure (Figures S4A and S4B). After 100 ns of simulation with DupA, Ub translocated and settled down in the catalytic pocket, whereas Ub did not find the catalytic pocket during the entire simulation (5 μs) with the SdeA PDE domain. Placing the PR-ubiquitinated peptide closer to the catalytic pocket of SdeA PDE domain resulted in a short residence time for both Ub and the peptide in the catalytic pocket, whereas the catalytic histidine of DupA remained close to the phosphate on the PR-ubiquitinated substrate throughout the simulation (5 μs) (Figures S4C and S4D). Based on these observations, we postulated that the differences in binding dynamics and affinities of DupA and SdeA to Ub might help explain how two similar PDE domains elicit two counteracting reactions. To explore this, we introduced multiple mutations in the DupA PDE domain (Figure 3F). Only the DupA E242R mutant affected the hydrolase ability and displayed reduced binding affinity to PR-ubiquitinated substrates (Figure 3G). More importantly, the same mutant was now able to promote stable PR ubiquitination (Figure 3H). These data reveal that the extent of interaction between PDE domains and Ub governs the directionality of their enzymatic activity. The hydrolase (PR deubiquitinase) activity is favored by high affinity and longer residence time of the PR-ubiquitinated substrate to DupA/B, while the transferase (PR-Ub ligase) activity is dictated by lower-affinity interactions of the SidE family PDE domain with ADPR-Ub. Until now, no general workflow for the specific enrichment of PR-ubiquitinated proteins has been presented. Initial concepts relied on the use of tagged Ub that lacks the C-terminal GG motif (UbΔGG) and can only be attached to other proteins by PR ubiquitination. However, this concept has several drawbacks and limitations, as it relies on overexpression of tagged Ub, which might stress the cell and is only usable in genetically engineered or transfected cells. Therefore, we aimed to establish a protocol that enables the efficient enrichment of PR-ubiquitinated proteins that do not rely on genetic perturbations. Given that DupA displayed strong binding affinity to PR-ubiquitinated substrates, we hypothesized that catalytic inactive mutants of DupA (H67A or H189N) could be used as trapping mutants to enrich PR-ubiquitinated proteins from cellular lysates for subsequent proteomic analysis. To test this, we first co-expressed SdeA together with HA-tagged Ub 1-74 (HA-UbΔGG), which lacks two glycine residues at the carboxy terminus, thereby preventing canonical ubiquitination. SdeA utilizes HA-UbΔGG to catalyze ubiquitination of cellular proteins, as demonstrated by a smear of PR-ubiquitinated proteins in cells (Figure 4A). Importantly, both DupA inactive mutants (H67A or DupA H189N) effectively bound and enriched PR-ubiquitinated substrates from cells co-expressing UbΔGG and WT SdeA, but not canonical ubiquitinated proteins from cells expressing mutant SdeA (H277A or EE/AA, Figures 4A and S5A). Interestingly, DupA H67A mutant enriched ADPR-Ub and ADPR-Ub-conjugated proteins from cells expressing SdeA (H277A), while H189N mutant could not. Moreover, the enriched PR-ubiquitinated substrates on DupA-trapping mutants could be cleaved in vitro by incubation with WT DupA (Figure S5B). Next, we sought to identify endogenous substrates in Legionella-infected cells. We first established and tested different Legionella strains for their ability to modulate PR-ubiquitinated substrates in infected cells. Infection with WT Legionella strain (Lp02) showed maximum PR ubiquitination of Rab33b at 2 h post-infection and subsequent reduction, while a strain lacking DupA (ΔdupA) maintained the PR ubiquitination up to 6 h post-infection (Figure 4B). Reconstitution of the ΔdupA strain with the DupA H67A mutant led to a slight increase in endogenous Rab33b PR ubiquitination , suggesting that DupA H67A acts as a dominant-negative mutant in infected cells. Moreover, deletion of DupB (ΔdupB) or both DupA and DupB (ΔdupA/B) led to an increased and more prolonged endogenous Rab33b PR ubiquitination 4–6 h post-infection (Figure S5C). This indicates that DupA and DupB may regulate PR ubiquitination at different stages of infection. Deletion of both DupA and DupB had no significant effect on overall Legionella proliferation in cultured cells (Figure S5D). In order to identify endogenously modified proteins, we used label-free MS quantification and examined the differential enrichment of PR-ubiquitinated proteins under the different infection conditions. We enriched PR-ubiquitinated proteins from cells infected with WT Legionella or the ΔdupA/B strains by GST-DupA H67A pull-down (Figure 4C). Eluted PR-ubiquitinated proteins released from the beads by cleaving the phosphodiester bond to the substrate serine using DupA (WT). Due to this non-denaturing elution strategy, only PR-ubiquitinated proteins and not unspecific binders, such as canonically ubiquitinated proteins, were eluted and further analyzed. We reproducibly quantified more than 1,000 proteins from cells infected with WT Legionella or the ΔdupA/B strain by using the DupA H67A-trapping mutant (Figures 4C and 4D; Table S2). Of these, 181 proteins were consistently and significantly enriched in cells infected with" @default.
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• W2983004433 creator A5007365210 @default.
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• W2983004433 date "2020-01-01" @default.
• W2983004433 modified "2023-10-17" @default.
• W2983004433 title "Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB" @default.
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• W2983004433 doi "https://doi.org/10.1016/j.molcel.2019.10.019" @default.
• W2983004433 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6941232" @default.
• W2983004433 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31732457" @default.
• W2983004433 hasPublicationYear "2020" @default.
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