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Ester-linked ubiquitination of serine or threonine residues - or even lipids - has emerged as a new regulatory earmark in cell signalling. Petrova et al. (2021) now reveal that ubiquitin esterification by the atypical ubiquitin ligase HOIL-1, a component of the LUBAC complex, is critical for proper formation of linear ubiquitin chains and control of immune signalling in T cells and macrophages. Surprisingly, ester-linked ubiquitination can either promote or inhibit linear ubiquitin conjugation and cytokine production depending on the receptor and immune cell engaged. Comment on: https://doi.org/10.1111/febs.15896.

The versatility of ubiquitination to impose control over vast domains of eukaryotic biology is due, in part, to diversification through differently-linked poly-ubiquitin chains. Deciphering the signaling roles for some poly-ubiquitin chain types, including those linked via K6, has been stymied by a lack of stringent linkage specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin, and in some cases even with exquisite specificity for distinct poly-ubiquitin signals. Herein, we identify and characterize a deubiquitinase domain of the secreted effector protein LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin during infection. We demonstrate the utility of LotA as a tool for studying K6 poly-ubiquitin. By determining apo and diUb-bound structures, we identify the mechanism of LotA activation and K6 poly-ubiquitin specificity, and identify a novel ubiquitin-binding domain utilized among bacterial deubiquitinases.

Ubiquitylation, a wide-spread post-translational protein modification in eukaryotes, marks cytosol‐invading bacteria as cargo for anti-bacterial autophagy.(1–3) The identity of the ubiquitylated substrate on bacteria has remained unknown. Here we show that the ubiquitin coat on cytosol-invading Salmonella is formed through the unprecedented ubiquitylation of a non-proteinaceous substrate, the lipid A moiety of bacterial lipopolysaccharide (LPS), by the E3 ubiquitin ligase RNF213. RNF213 is a risk factor for Moyamoya disease (MMD)(4,5), a progressive stenosis of the supraclinoid internal carotid artery that causes stroke, especially in children.(6,7) RNF213 restricts the proliferation of cytosolic Salmonella and is essential for the generation of the bacterial ubiquitin coat, both directly, through ubiquitylation of LPS, and indirectly, through recruitment of LUBAC, a downstream E3 ligase that adds M1-linked ubiquitin chains onto pre-existing ubiquitin coats.(8) In cells lacking RNF213 bacteria do not attract ubiquitin-dependent autophagy cargo receptors and fail to induce anti-bacterial autophagy. The ubiquitylation of LPS on cytosol-invading Salmonella requires the dynein-like core of RNF213 but not its RING domain. Instead, LPS ubiquitylation relies on an RZ finger in the E3 shell. We conclude that ubiquitylation extends beyond protein substrates, that LPS ubiquitylation triggers cell-autonomous immunity and we postulate that non- proteinaceous substances other than LPS may also become ubiquitylated.

A new study reveals that, in addition to its longstanding role in recruiting proteins to the proteasome, ubiquitination can also induce a structural destabilization that allows the target protein to be efficiently unraveled for degradation.

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., Prediction and validation of pathogenic bacterial DUBs and insights into their structures and (poly)ubiquitin linkage specificities allows for first cross‐kingdom comparison of the OTU deubiquitination enzyme fold.

The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.

Summary 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., Graphical Abstract, Highlights • Bacterial CE proteases exhibit distinct ubiquitin/ubiquitin-like specificities • Substrate specificity is acquired through variability in three common regions • Structural and functional data redefine CE clan relationships across kingdoms • CE effectors are fitted with accessory domains that modulate function, Focusing on examples from pathogenic bacteria, Pruneda et al. examine a family of proteases that displays remarkably distinct specificities toward ubiquitin and ubiquitin-like modifications. Leveraging structural and functional data, the authors derive mechanisms through which substrate specificity is achieved and redefine relationships within the enzyme family across kingdoms of life.

Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner.

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.

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.

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

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 Cys-73, His-162, and Asp-182. 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.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Summary Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed., Author Summary Many bacterial pathogens affecting humans, animals and plants export diverse proteins across the cell membranes into the medium surrounding the bacteria. Some of these secreted proteins are involved in pathogenesis. One example is cholera toxin secreted by the bacterium Vibrio cholerae, a causative agent of cholera. The sophisticated type II secretion system is responsible for moving this toxin, and several other proteins, across the outer membrane. Here, we studied the interaction between the outer membrane pore of the type II secretion system, the secretin GspD, and the inner membrane protein GspC. We have solved three crystal structures of complexes between the interacting domains and identified critical contacts in the GspC–GspD interface. We also showed the importance of these contacts for assembly of the secretion system and for secretion of proteins by V. cholerae. Our studies provide a major piece in the puzzle of how the type II secretion system is assembled and how it functions. One day this knowledge might allow us to design compounds which interfere with this secretion process. Such compounds would be useful in the battle against bacteria affecting human health.