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• W3035797211 abstract "Article22 June 2020Open Access Transparent process Identification and characterization of diverse OTU deubiquitinases in bacteria Alexander F Schubert orcid.org/0000-0002-1834-3787 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Justine V Nguyen orcid.org/0000-0001-9739-5261 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Tyler G Franklin orcid.org/0000-0002-0311-1190 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Paul P Geurink orcid.org/0000-0003-1849-1111 Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands Search for more papers by this author Cameron G Roberts orcid.org/0000-0002-0115-7013 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Daniel J Sanderson orcid.org/0000-0002-0516-814X Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Lauren N Miller orcid.org/0000-0001-6982-5041 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Huib Ovaa orcid.org/0000-0002-0068-054X Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands Search for more papers by this author Kay Hofmann orcid.org/0000-0002-2289-9083 Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Jonathan N Pruneda Corresponding Author [email protected] orcid.org/0000-0002-0304-4418 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author David Komander Corresponding Author [email protected] orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Department of Medical Biology, The University of Melbourne, Melbourne, VIC, Australia Search for more papers by this author Alexander F Schubert orcid.org/0000-0002-1834-3787 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Justine V Nguyen orcid.org/0000-0001-9739-5261 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Tyler G Franklin orcid.org/0000-0002-0311-1190 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Paul P Geurink orcid.org/0000-0003-1849-1111 Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands Search for more papers by this author Cameron G Roberts orcid.org/0000-0002-0115-7013 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Daniel J Sanderson orcid.org/0000-0002-0516-814X Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Lauren N Miller orcid.org/0000-0001-6982-5041 Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Huib Ovaa orcid.org/0000-0002-0068-054X Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands Search for more papers by this author Kay Hofmann orcid.org/0000-0002-2289-9083 Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Jonathan N Pruneda Corresponding Author [email protected] orcid.org/0000-0002-0304-4418 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author David Komander Corresponding Author [email protected] orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia Department of Medical Biology, The University of Melbourne, Melbourne, VIC, Australia Search for more papers by this author Author Information Alexander F Schubert1,†,‡, Justine V Nguyen2,‡, Tyler G Franklin2, Paul P Geurink3, Cameron G Roberts2, Daniel J Sanderson2, Lauren N Miller2, Huib Ovaa3, Kay Hofmann4, Jonathan N Pruneda *,1,2 and David Komander *,1,5,6 1Medical Research Council Laboratory of Molecular Biology, Cambridge, UK 2Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA 3Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Centre, Leiden, The Netherlands 4Institute for Genetics, University of Cologne, Cologne, Germany 5Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 6Department of Medical Biology, The University of Melbourne, Melbourne, VIC, Australia †Present address: Department of Structural Biology, Genentech Inc., South San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: 1 (503) 494 8102; E-mail: [email protected] *Corresponding author. Tel: +61 3 9345 2670; E-mail: [email protected] EMBO J (2020)39:e105127https://doi.org/10.15252/embj.2020105127 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases. Synopsis OTU-family deubiquitinases (DUBs) regulate human ubiquitin signaling and have been subverted by viruses, while their use by pathogenic bacteria remains less-well characterized. Broader identification and characterization of bacterial OTU DUBs now allows for first cross-kingdom comparison of this deubiquitination enzyme fold. Bioinformatic prediction identifies eukaryote-like OTU deubiquitinase domains across a broad set of bacterial species. Eight predicted bacterial OTU DUBs were recombinantly expressed and biochemically validated. Enzymology and crystal structures reveal novel ubiquitin interaction modes. An alternate direction of sequence threading through the OTU fold exemplifies a tolerance to permutation. Cross-kingdom structural analysis of the OTU deubiquitinase fold allows categorizing the variable structural motifs responsible for interactions with substrate ubiquitin. Introduction Outside of its canonical role in targeted proteasomal degradation, ubiquitin (Ub) signaling plays crucial roles in many other aspects of eukaryotic biology, including immune responses (Swatek & Komander, 2016; Ebner et al, 2017). In fact, the ability of Ub modifications to form discrete polymers (polyUb) allows it to perform multiple signaling functions even within the same pathway (Komander & Rape, 2012). TNF signaling, for example, relies upon the concerted action of several nondegradative polyUb signals (K63-, M1-, and K11-linked chains) as well as the degradative K48-linked chains in order to ultimately achieve NF-κB transcriptional activation (Ebner et al, 2017). PolyUb chains can also be combined into complex higher-order architectures that further diversify their signaling capacities (Haakonsen & Rape, 2019). These processes are tightly regulated by Ub ligases that assemble the signals, Ub-binding domains that respond to them, and specialized proteases termed deubiquitinases (DUBs) that remove them. Breakdown of this regulation can lead to immune hyper- or hypoactivation, and has been linked to several human diseases (Popovic et al, 2014). Although the Ub system is largely exclusive to eukaryotes, invading viruses and bacteria have evolved strategies for manipulating host Ub signaling responses during infection (Wimmer & Schreiner, 2015; Lin & Machner, 2017). These strategies can include pathogen-encoded Ub ligases or DUBs that redirect or remove host signals, respectively. Pathogen-encoded DUBs can affect host functions such as innate immune activation, autophagy, or morphology (Mesquita et al, 2012; Pruneda et al, 2018; Wan et al, 2019). When their ability to remove host Ub signals is taken away, some pathogens show reduced fitness and infectivity (Rytkönen et al, 2007; Fischer et al, 2017). Interestingly, though some bacterial DUBs are entirely foreign and reflect convergent evolution (Wan et al, 2019), others appear to adopt eukaryote-like protein folds and/or mechanisms (Pruneda et al, 2016). Humans encode six families of cysteine-dependent DUBs that all fall underneath the CA clan of proteases and one family of Ub-specific metalloproteases from the MP clan. An additional family of ubiquitin-like proteases (ULPs) regulates NEDD8 and SUMO signaling and belongs to the CE cysteine protease clan. The majority of bacterial DUBs studied to date are related to the CE clan of ULPs and appear to predominantely target host K63-linked polyUb signals (Pruneda et al, 2016). The ULP fold is also widely used among viruses, both as a Ub-specific protease and as a traditional peptidase (Wimmer & Schreiner, 2015). Another DUB fold that is common to both eukaryotes and viruses is the ovarian tumor (OTU) family. Humans encode 16 DUBs of the OTU family with important functions in signaling pathways such as innate immunity and cell cycle regulation (Du et al, 2019). Some OTUs, such as OTUB1 and OTULIN, are highly specific for certain polyUb signals (K48- and M1-linked chains, respectively), and these properties not only provide insight into their biological functions (proteasomal degradation and inflammatory signaling, respectively), but also prove useful for technological applications such as ubiquitin chain restriction analysis (Keusekotten et al, 2013; Mevissen et al, 2013; Du et al, 2019). Viruses use OTU DUBs to block innate immune activation during infection, often by cleaving both Ub and the antiviral Ub-like modifier ISG15 (Bailey-Elkin et al, 2014). In bacteria, however, only two reported cases of the OTU fold have been identified. The first, ChlaOTU from Chlamydia pneumoniae, was predicted by sequence similarity (Makarova et al, 2000) and shown to play an active role in the clearance of Ub signals following infection (Furtado et al, 2013). The second example, LotA, plays a similar role in Legionella pneumophila infection (Kubori et al, 2018). Whether these bacterial OTUs were unique, however, or represent a wider adaptation of the OTU fold among bacteria remained unknown. To determine whether, like the CE clan ULPs, the OTU fold is a common adaptation for DUB activity across bacteria, we generated an OTU sequence profile and predicted distantly related examples among bacterial genomes. Using an array of Ub substrates and in vitro assays, we confirmed that predicted OTUs from pathogens such as Escherichia albertii, L. pneumophila, and Wolbachia pipientis were bona fide DUBs. Furthermore, with one exception all of our confirmed OTUs were Ub-specific (over Ub-like modifiers) and targeted a defined subset of polyUb chain types, much like human OTUs (Mevissen et al, 2013). Structural analysis of two examples revealed novel modes of Ub substrate recognition and, surprisingly, even a permutated sequence topology that still gives rise to a familiar OTU fold. Our new bacterial OTU DUB structures allowed for the first cross-kingdom structural analysis, from which we established a framework for identifying evolutionary adaptations in the S1 substrate-binding site that impart DUB activity. This work establishes the OTU fold as a common tool used by bacteria to manipulate host Ub signaling and provides insight into the origins and adaptations of the OTU fold across eukaryotes, bacteria, and viruses. Results Identification of bacterially encoded OTU deubiquitinases Given the expansive use of the OTU DUB fold in eukaryotes and viruses to regulate key aspects of cellular biology and infection, respectively (Du et al, 2019), we sought to determine whether, like the CE clan ULPs (Pruneda et al, 2016), the family extends into bacteria as well. Through generating a sequence alignment of eukaryotic and viral OTU domains, we created a generalized sequence profile that was used to identify related sequences among bacteria. Candidates identified through this approach were further scrutinized by secondary structure prediction and domain recognition using the Phyre2 server (Kelley et al, 2015). Those that encoded active site sequences matching the Pfam motif (Pfam Entry PF02338) embedded within appropriate elements of secondary structure (e.g., an active site Cys motif at the beginning of an α-helix) were prioritized for subsequent validation (Fig EV1A). Reassuringly, this approach also detected the first characterized bacterial OTU, ChlaOTU (Makarova et al, 2000; Furtado et al, 2013), and we followed this naming convention for predictions with previously unknown function. Despite encoding two OTU domains (Kubori et al, 2018), LotA was not detected by our approach. For biochemical validation, we selected E. albertii “EschOTU” (GenBank EDS93808.1), L. pneumophila ceg7 (lpg0227, GenBank AAU26334.1), Burkholderia ambifaria “BurkOTU” (GenBank EDT05193.1), C. pneumoniae ChlaOTU (CPn_0483, GenBank AAD18623.1), Rickettsia massiliae “RickOTU” (dnaE2, GenBank ABV84894.1), W. pipientis strain wPip “wPipOTU” (WP0514, GenBank CAQ54622.1), W. pipientis wMel “wMelOTU” (WD_0443, GenBank AAS14166.1), and L. pneumophila ceg23 (lpg1621, GenBank AAU27701.1) (Fig 1A and B). Click here to expand this figure. Figure EV1. Prediction and validation of OTU DUBs from bacteria A. Workflow illustrating the process used for bioinformatic prediction of bacterial OTU domains, followed by their manual curation. Representative output from the Phyre2 curation is shown for the predicted EschOTU active site. B. Table presenting prediction scores for type III and type IV secretion signals using the pEFFECT (Goldberg et al, 2016) and S4TE 2.0 (Noroy et al, 2019) prediction approaches, respectively. For pEFFECT predictions, prediction reliability scores above 50 or above 80 are associated with 87% or 96% accuracy, respectively. For S4TE 2.0, a prediction score above 72 is associated with a 98% sensitivity. Organisms that lack one of the secretion systems are marked as not applicable (NA). C. Full fluorescent Ub substrate cleavage data for all bacterial OTUs following Ala substitution at each member of the predicted catalytic triad. These data were collected in parallel with those presented in Fig 1G, and the WT dataset is shown again for clarity. The rise above 100% observed with BurkOTU is indicative of a noncovalent interaction. Download figure Download PowerPoint Figure 1. Prediction and validation of OTU DUBs from bacteria A. Pfam-generated sequence logo of the regions surrounding the OTU catalytic Cys and general base His (marked with asterisks). The conservation of these regions in the human OTUB1 and predicted bacterial OTUs are shown below, together with their relative order in the sequence topology indicated by the sequence position as well as green and red arrows for the typical and atypical arrangements, respectively. B. Bacterial species to which the predicted OTUs belong. C. Outcome of interactions between the highlighted bacterial species and their respective eukaryotic hosts. D. Percent identity matrix calculated from a PSI-Coffee alignment (Notredame et al, 2000) of the predicted OTU domains. OTUB1 (80–271), EschOTU (184–362), ceg7 (1–298), BurkOTU (186–315), ChlaOTU (193–473), RickOTU (161–356), wPipOTU (66–354), wMelOTU (40–205), and ceg23 (9–277) were used to create the alignment. E. Coomassie-stained SDS–PAGE gel showing purified protein from the predicted bacterial OTU constructs. F. Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks. G. Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding. H. Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in (G), including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants. Download figure Download PowerPoint Our selected candidates are encoded by a wide range of Gram-negative bacteria that span the chlamydiae, alpha-, beta-, and gammaproteobacterial classes (Fig 1B). Consistent with putative host-targeted DUB activity, all of the identified species have reported interactions with eukaryotic hosts (Fig 1C), some of which are linked to severe human diseases (e.g., Legionnaire's disease) or altered biology (e.g., Wolbachia sex determination). The majority of our candidates arise from obligate intracellular bacteria that depend upon host interactions for survival. All of the selected bacterial OTU-containing proteins were predicted by pEFFECT or S4TE 2.0 to also encode either type III or type IV secretion signals (Goldberg et al, 2016; Noroy et al, 2019), suggesting potential roles as secreted effectors (Fig EV1B). With the exception of ChlaOTU, which had no recognizable conservation of the general base His motif, all of the selected examples contained both catalytic Cys and general base His consensus sequences that closely matched the established motifs and secondary structure of OTUs (Fig 1A). Remarkably, however, our active site analysis suggested that some examples, particularly EschOTU, could thread through the OTU fold in a topology that is distinct from any previously studied example (Fig 1A, red arrow). Outside of the active site motifs, our OTU domain predictions have strikingly low sequence similarity to each other and to the archetypal human example, OTUB1, that centers around only ~ 15% identity (Fig 1D). To test our predictions for DUB activity, we synthesized coding regions or amplified them from bacterial samples, designed constructs that (where possible) contain the minimal predicted OTU domain, and proceeded with Escherichia coli expression and purification (Fig 1E). We found the Legionella ceg7 protein to be the most difficult to work with, and after much effort arrived at a preparation that retained a SUMO solubility tag (Fig 1E). As a first measure of in vitro DUB activity, we treated the putative bacterial OTUs with a Ub-Propargylamine (Ub-PA) activity-based probe that covalently reacts with a DUB's active site Cys, resulting in an 8.5 kDa shift in molecular weight on SDS–PAGE (Ekkebus et al, 2013). By this approach, EschOTU, ceg7, BurkOTU, wMelOTU, and ceg23 all showed robust reactivity with the Ub-PA probe that was abolished following mutation of the predicted active site Cys to Ala (Fig 1F). This assay validated some of our OTU predictions and our identification of a catalytic Cys. To visualize genuine protease activity with improved sensitivity, we implemented a fluorescence polarization assay that detects the release of a C-terminal isopeptide-linked fluorescent peptide (Geurink et al, 2012). In addition to EschOTU, ceg7, wMelOTU, and ceg23, this assay could also detect DUB activity for RickOTU (albeit at high enzyme concentration) (Fig 1G). ChlaOTU, wPipOTU, and BurkOTU showed no activity against this substrate, but BurkOTU did exhibit a dramatic increase in fluorescence polarization indicative of a strong interaction with the Ub substrate (Fig 1G). The observation of binding without cleavage could indicate that either BurkOTU has a high-affinity Ub binding site outside of the S1 site or the orientation of the catalytic site may be regulated through some other means. For those that demonstrated activity against the fluorescent Ub substrate, we additionally tested for dependence upon our predicted active site triad residues (catalytic Cys, general base His, and acidic). In all cases, mutation of the Cys or His residues to Ala abolished DUB activity (Figs 1H and EV1C). The acidic position is typically the second amino acid C-terminal to the general base His, and in similar manner to human OTUs, its mutation can result in complete, intermediate, or no loss of activity in the bacterial OTUs (Figs 1H and EV1C). Members in the A20 subfamily of human OTUs encode their acidic residue N-terminal to the catalytic Cys (Komander & Barford, 2008); we predicted a similarly positioned acidic residue in the ceg23 sequence (D21), and its mutation abolished DUB activity (Figs 1H and EV1C). Substrate specificities of bacterial OTU deubiquitinases Across eukaryotic and viral examples, the OTU family has been shown to display a remarkable diversity in substrates specificities, both at the level of Ub/Ub-like specificity (e.g., Crimean Congo hemorrhagic fever virus vOTU dual Ub/ISG15 activity (Frias-Staheli et al, 2007; Akutsu et al, 2011; James et al, 2011)) and at the level of polyUb chain types [e.g., K11, K48, or M1 specificity (Mevissen et al, 2013)]. Therefore, we sought to assess our bacterial OTUs for both types of substrate specificity. To measure Ub/Ub-like specificity, we used fluorescence polarization to measure activity toward Ub, ISG15, NEDD8, and SUMO1 in parallel (Figs 2A–C and EV2A). EschOTU, ceg7, RickOTU, and wMelOTU primarily targeted Ub under these conditions (Figs 2A and C, and EV2A). In addition to its activity toward the Ub substrate, ceg23 could also cleave the SUMO1 substrate (Figs 2C and EV2A). This particular combination of Ub/Ub-like proteolytic activities had previously only been observed in XopD from the plant pathogen Xanthomonas campestris (Pruneda et al, 2016). While BurkOTU did not demonstrate any cleavage of the Ub/Ub-like substrates, the increased signal indicative of an interaction with the Ub substrate was specific and was not observed with any of the Ub-like substrates (Fig 2B and C). ChlaOTU and wPipOTU showed no activity against any of the Ub/Ub-like substrates. Figure 2. Substrate specificity profiling of bacterial OTU DUBs A. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. B. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate. C. Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration. D. Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS–PAGE, and visualized by Coomassie staining. E. Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS–PAGE, and visualized by Coomassie staining. F. Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Substrate specificity profiling of bacterial OTU DUBs A. Corrected Ub/Ub-like substrate specificity assays for all bacterial OTUs. B. Ub chain specificity assays for EschOTU, ceg7, ChlaOTU, RickOTU, wPipOTU, and ceg23 toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS–PAGE, and visualized by Coomassie staining. C. K11 diUb cleavage assay for BurkOTU WT and Ala-substituted catalytic triad mutants. Reaction samples were quenched at the indicated timepoints, resolved by SDS–PAGE, and visualized by Coomassie staining. Download figure Download PowerPoint Specificity at the level of polyUb chain type was measured by constructing a panel of all eight canonical diUb linkages for use in gel-based cleavage assays (Mevissen et al, 2013; Michel et al, 2018). To better visualize any discrimination between chain types, enzyme concentration and incubation times were optimized such that at least one diUb species was nearly or completely cleaved by the end of the experiment (Figs 2D and E, and EV2B). Under no conditions were we able to observe activity for ChlaOTU or wPipOTU. All other bacterial OTUs (including BurkOTU) showed DUB activities with moderate discrimination between chain types (Fig 2F). Interestingly, EschOTU, ceg7, BurkOTU, RickOTU, wMelOTU, and ceg23 all shared a common basal preference for K6-, K11-, K48-, and K63-linked chains (Fig 2F), a combination not observed in any of the human OTU DUBs (Mevissen et al, 2013) but surprisingly similar to some viral OTUs (Dzimianski et al, 2019). Among these chain types, there were some indications of further preference: EschOTU, ceg7, and RickOTU demonstrated a slight preference toward K48-linked chains, BurkOTU toward K11, wMelOTU toward K6, and ceg23 more strongly toward K63 linkages (Figs 2D–F and EV2B). Underneath these preferences were several lowly cleaved background activities, including K33-linked chains across all active examples and an additional activity toward M1-linked chains from ceg7. Notably, aside from reactivity with the Ub-PA probe, diUb cleavage offered the first robust measure of activity for BurkOTU and allowed for the confirmation of all three predicted active site triad residues by mutagenesis (Fig EV2C). The peculiar requirement of polyUb chains for BurkOTU activity is reminiscent of OTULIN (Keusekotten et al, 2013) and could indicate a mechanism by which binding to the S1’ site drives substrate recognition and catalysis. Bacterial OTU deubiquitinases demonstrate novel modes of substrate recognition To confirm that our validated bacterial DUBs are indeed members of the OTU family, we determined a crystal structure of wMelOTU to 1.5 Å resolution by molecular replacement with the core structure of yeast OTU1 (Messick et al, 2008; Figs 3A and EV3A, Table 1). The wMelOTU structure exhibits a pared-down canonical OTU domain architecture with a central β-sheet supported underneath by an α-helical subdomain, but although additional α-helical content typically sandwiches the β-sheet from above, there is very little additional support in the wMelOTU structure (Figs 3A and B, and EV3B). The core of the OTU fold that contains the active site (the central β-sheet and two most proximal supporting α-helices) closely resembles other OTU domains such as OTUB1 (Fig 3B, 1.6 Å RMSD) and vOTU (Fig EV3B, 1 Å RMSD), whereas the surrounding areas of structure are more divergent (Akutsu et al, 2011; Juang et al, 2012). Two regions of structure near the S1 substrate recognition site, encompassing 6 and 7 amino acids, respectively, are missing from the electron density (Figs 3A and EV3A). The structure confirms our prediction and mutagenesis of active site residues (Figs 1A and H, and 3A). However, the catalytic triad is misaligned (Fig 3A) as a result of the loop preceding the general base His (the so-called His-loop) occupying a descended conformation that would also occlude entry of the Ub C-terminus into the active site (Fig 3C). Thus, while the apo wMelOTU structure validates our prediction of an OTU fold, it raised new questions as to the mechanisms of substrate recognition. Figure 3. wMelOTU structure reveals novel Ub embrace mechanism A. Cartoon representation of the 1.5 Å Wolbachia pipientis wMelOTU crystal structure with labeled termini, missing regions," @default.
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• W3035797211 title "Identification and characterization of diverse OTU deubiquitinases in bacteria" @default.
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