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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification and characterization of diverse OTU deubiquitinases in bacteria Alexander F. Schubert , Justine V Nguyen , Tyler G. Franklin , Paul P. Geurink , Cameron G. Roberts , Daniel J. Sanderson , Lauren N. Miller , Huib Ovaa , Kay Hofmann , Jonathan N. Pruneda , David Komander The Embo Journal 81 DOI: 10.15252/embj.2020105127 Diamond Proposal Number(s): [8547, 11235] Open Access Show Abstract ▼ 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.	Jun 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A Chlamydia effector combining deubiquitination and acetylation activities induces Golgi fragmentation Jonathan N. Pruneda , Robert J. Bastidas , Erithelgi Bertsoulaki , Kirby N. Swatek , Balaji Santhanam , Michael J. Clague , Raphael H. Valdivia , Sylvie Urbé , David Komander Nature Microbiology 130 DOI: 10.1038/s41564-018-0271-y Diamond Proposal Number(s): [8547, 11235] Show Abstract ▼ Abstract: Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.	Nov 2018
I04-1-Macromolecular Crystallography (fixed wavelength)	Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies Kirby N. Swatek , Martina Aumayr , Jonathan N. Pruneda , Linda J. Visser , Stephen Berryman , Anja F. Kueck , Paul P. Geurink , Huib Ovaa , Frank J. M. Van Kuppeveld , Tobias J. Tuthill , Tim Skern , David Komander Proceedings Of The National Academy Of Sciences 35 DOI: 10.1073/pnas.1710617115 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.	Feb 2018
I04-Macromolecular Crystallography	An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1 Christina Gladkova , Alexander F. Schubert , James Wagstaff , Jonathan N. Pruneda , Stefan M. V. Freund , David Komander The Embo Journal DOI: 10.15252/embj.201797876 Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.	Nov 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	The Salmonella Effector SpvD is a Cysteine Hydrolase with a Serovar-Specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses and Bacterial Virulence Grzegorz J. Grabe , Yue Zhang , Michal Przydacz , Nathalie Rolhion , Yi Yang , Jonathan N. Pruneda , David Komander , David W. Holden , Stephen A. Hare Journal Of Biological Chemistry DOI: 10.1074/jbc.M116.752782 Diamond Proposal Number(s): [9424] Open Access Show Abstract ▼ Abstract: Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising C73, H162, and D182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro. A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.	Oct 2016
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases Jonathan N. Pruneda , Charlotte h. Durkin , Paul p. Geurink , Huib Ovaa , Balaji Santhanam , David w. Holden , David Komander Molecular Cell 63, 261 - 276 DOI: 10.1016/j.molcel.2016.06.015 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins.	Jul 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes Monique P. C. Mulder , Katharina Witting , Ilana Berlin , Jonathan N. Pruneda , Kuen-phon Wu , Jer-gung Chang , Remco Merkx , Johanna Bialas , Marcus Groettrup , Alfred C. O. Vertegaal , Brenda A Schulman , David Komander , Jacques Neefjes , Farid El Oualid , Huib Ovaa Nature Chemical Biology 12, 523 - 530 DOI: 10.1038/nchembio.2084 Diamond Proposal Number(s): [11235] Show Abstract ▼ Abstract: Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.	May 2016
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Assembly and Specific Recognition of K29- and K33-Linked Polyubiquitin Martin a Michel , Paul r Elliott , Kirby n. Swatek , Michal Simicek , Jonathan Pruneda , Jane l. Wagstaff , Stefan m.v. Freund , David Komander Molecular Cell DOI: 10.1016/j.molcel.2015.01.042 PMID: 25752577 Diamond Proposal Number(s): [8547] Open Access Show Abstract ▼ Abstract: Protein ubiquitination regulates many cellular processes via attachment of structurally and functionally distinct ubiquitin (Ub) chains. Several atypical chain types have remained poorly characterized because the enzymes mediating their assembly and receptors with specific binding properties have been elusive. We found that the human HECT E3 ligases UBE3C and AREL1 assemble K48/K29- and K11/K33-linked Ub chains, respectively, and can be used in combination with DUBs to generate K29- and K33-linked chains for biochemical and structural analyses. Solution studies indicate that both chains adopt open and dynamic conformations. We further show that the N-terminal Npl4-like zinc finger (NZF1) domain of the K29/K33-specific deubiquitinase TRABID specifically binds K29/K33-linked diUb, and a crystal structure of this complex explains TRABID specificity and suggests a model for chain binding by TRABID. Our work uncovers linkage-specific components in the Ub system for atypical K29- and K33-linked Ub chains, providing tools to further understand these unstudied posttranslational modifications.	Mar 2015
I03-Macromolecular Crystallography	Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis Tobias Wauer , K. N. Swatek , J. L. Wagstaff , C. Gladkova , Jonathan Pruneda , M. A. Michel , Malte Matthias Gersch , C. M. Johnson , S. M. Freund , David Komander The Embo Journal 34, 307 - 325 DOI: 10.15252/embj.201489847 PMID: 25527291 Diamond Proposal Number(s): [8547] Open Access Show Abstract ▼ Abstract: Ubiquitin phosphorylation by PINK1 kinase is involved in Parkin activation. Structural and biochemical work now shows that it also affects ubiquitin conformation, chain assembly and deconjugation, thus potentially affecting ubiquitin signaling more broadly.	Feb 2015

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