Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N-terminal portion which comprises a canonical RING domain, one or two B-box domains and a coiled-coil region that mediates ligase dimerization. Self-association via the coiled-coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin-loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full-length protein. Our data reveal an unexpected diversity in the self-association mechanism of TRIMs that might be crucial for their biological function.

Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases.

TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.

Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.

Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.

Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP.

Linear ubiquitin chains are important regulators of cellular signalling pathways that control innate immunity and inflammation through nuclear factor (NF)-κB activation and protection against tumour necrosis factor-α-induced apoptosis. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multisubunit E3 ligase. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or 'donor' ubiquitin. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The carboxy-terminal portion of HOIP adopts a novel fold that, together with a zinc-finger, forms a ubiquitin-binding platform that orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3∼ubiquitin thioester. The C-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine, providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-κB pathway in vivo, and they explain the determinants of linear ubiquitin chain specificity by LUBAC.

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response.

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.

Structural determinants of TRIM protein function.

Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of Really Interesting New Gene (RING) E3 ubiquitin ligases and contribute to the regulation of numerous cellular activities, including innate immune responses. The conserved TRIM harbours a RING domain that imparts E3 ligase activity to TRIM family proteins, whilst a variable C-terminal region can mediate recognition of substrate proteins. The knowledge of the structure of these multidomain proteins and the functional interplay between their constituent domains is paramount to understanding their cellular roles. To date, available structural information on TRIM proteins is still largely restricted to subdomains of many TRIMs in isolation. Nevertheless, applying a combination of structural, biophysical and biochemical approaches has recently allowed important progress to be made towards providing a better understanding of the molecular features that underlie the function of TRIM family proteins and has uncovered an unexpected diversity in the link between self-association and catalytic activity.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

Mechanism and function of Vav1 localisation in TCR signalling.

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate.

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP--the catalytic subunit of LUBAC--is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.

Covalent fragment-based drug discovery for target tractability.

An important consideration in drug discovery is the prioritization of tractable protein targets that are not only amenable to binding small molecules, but also alter disease biology in response to small molecule binding. Covalent fragment-based drug discovery has emerged as a powerful approach to aid in the identification of such protein targets. The application of irreversible binding mechanisms enables the identification of fragment hits for challenging-to-target proteins, allows proteome-wide screening in a cellular context, and makes it possible to determine functional effects with modestly potent ligands without the requirement for extensive compound optimization. Here, we provide an overview of recent approaches to covalent fragment-based screening and discuss how these have been applied to establish the tractability of unexplored binding sites on protein targets.

Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold.

SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.

It's a TRIM-endous view from the top: the varied roles of TRIpartite Motif proteins in brain development and disease.

The tripartite motif (TRIM) protein family members have been implicated in a multitude of physiologies and pathologies in different tissues. With diverse functions in cellular processes including regulation of signaling pathways, protein degradation, and transcriptional control, the impact of TRIM dysregulation can be multifaceted and complex. Here, we focus on the cellular and molecular roles of TRIMs identified in the brain in the context of a selection of pathologies including cancer and neurodegeneration. By examining each disease in parallel with described roles in brain development, we aim to highlight fundamental common mechanisms employed by TRIM proteins and identify opportunities for therapeutic intervention.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments.

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.

Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization.

An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 61.55, c = 222.81 Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100 K to 2.6 and 2.0 Šresolution, respectively.