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TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1. NS1 of influenza A virus inhibits TRIM25 activity, which is an E3 ligase important for induction of the interferon response. Here, Koliopoulos et al. present structures of TRIM25 and NS1 and show how NS1 binding interferes with substrate recognition of TRIM25.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair. The authors present the full-length dimeric TRIM72 E3 ubiquitin ligase and the architecture of its high-order assembly bound to a phosphatidylserine-enriched membrane, providing insights into its role in membrane repair and ubiquitylation.

The exposed N-terminal or C-terminal residues of proteins can act, in cognate sequence contexts, as degradation signals (degrons) that are targeted by specific E3 ubiquitin ligases for proteasome-dependent degradation by N-degron or C-degron pathways. Here, we discovered a distinct C-degron pathway, termed the Gln/C-degron pathway, in which the B30.2 domain of E3 ubiquitin ligase TRIM7 (TRIM7B30.2) mediates the recognition of proteins bearing a C-terminal glutamine. By determining crystal structures of TRIM7B30.2 in complexes with various peptides, we show that TRIM7B30.2 forms a positively charged binding pocket to engage the “U”-shaped Gln/C-degron. The four C-terminal residues of a substrate play an important role in C-degron recognition, with C-terminal glutamine as the principal determinant. In vitro biochemical and cellular experiments were used to further analyze the substrate specificity and selective degradation of the Gln/C-degron by TRIM7.

The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α. Tripartite-motif containing (TRIM) proteins modulate cellular responses to viral infection. Here the authors use molecular dynamics simulations to demonstrate that TRIM5α uses a two-dimensional lattice hopping mechanism to aggregate on the HIV capsid surface and initiate lattice growth.

TRIpartite Motif (TRIM) family proteins have diverse roles across a broad variety of cellular functions, which are largely presumed to depend on their ubiquitin E3 ligase activity, conferred by a RING domain. However, recent reports have shown that some TRIMs lack detectable ubiquitination activity in isolation, despite containing a RING domain. Here, we present parallel in cellulo, in vitro, and in silico structure-function analyses of the ubiquitin E3 ligase activity and RING domain structural characteristics of the whole TRIM protein family. In-depth follow-up studies of this comprehensive dataset reveals a number of ‘pseudoligases’, whose RING domains have structurally diverged at either the homodimerisation or E2~ubiquitin interfaces, thereby disrupting their ability to catalyse ubiquitin transfer. Together, these data raise intriguing open questions regarding the unknown TRIM functions in physiology and disease. Understanding the catalytic and regulatory mechanisms of the ubiquitin system is key to tapping into its therapeutic potential in diseases where its dysfunction is implicated. Here, the authors uncover a set of catalytically dead ‘pseudo’ ubiquitin ligases with possible regulatory roles.

Breast cancer is the most common cancer in the world. Relapse and metastasis are important factors endangering the life of breast cancer patients, but the mechanism is still unclear. The stabilization of p53 is essential for preventing carcinogenesis, and ubiquitination is one of the main ways to regulate the stability of p53. Tripartite motif-containing 31 (TRIM31) is a new member of the TRIM family and functions as an E3 ubiquitin ligase. It acts as a cancer promoter or suppressor in the malignant processes of multiple cancers. However, the function of TRIM31 in breast cancer progression remains unknown. In this study, we showed that TRIM31 is downregulated in breast cancer tissues and negatively correlated with breast cancer progression. Both gain- and loss-of-function assays indicated that TRIM31 inhibits the proliferation, colony formation, migration, and invasion of breast cancer cells. Further investigation demonstrated that TRIM31 directly interacts with p53, and inducing the K63-linked ubiquitination of p53 via its RING domain, Meanwhile, TRIM31 suppresses the MDM2-mediated K48-linked ubiquitination of p53 through competitive inhibiting the interaction of MDM2 and p53, leading to the p53 stabilization and activation. Knockdown of p53 reversed the inhibitory effects of TRIM31 on the growth and metastasis of breast cancer cells. Moreover, we found that the RING and coiled-coil (C–C) domains of TRIM31 were essential for its tumor suppressor function. Taken together, our findings reveal a novel mechanism by which TRIM31 suppresses breast cancer development through the stabilization and activation of p53 and define a promising therapeutic strategy for restoring TRIM31 to treat breast cancer.

ZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity. We show that KHNYN is an active nuclease that acts in a partly redundant manner with its homolog N4BP1. The ZAP N-terminal RNA binding domain constitutes a minimal core that is essential for antiviral complex activity, and we present a crystal structure of this domain that reveals contacts with the functionally required KHNYN C-terminal domain. These contacts are remote from the ZAP CpG binding site and would not interfere with RNA binding. Based on our dissection of ZAP, TRIM25 and KHNYN functional anatomy, we could design artificial chimeric antiviral proteins that reconstitute the antiviral function of the intact authentic proteins, but in the absence of protein domains that are otherwise required for activity. Together, these results suggest a model for the RNA recognition and action of ZAP-containing antiviral protein complexes. Structural and functional studies of Zinc finger antiviral protein and its cofactors, TRIM25 and KHYNYN, reveal the formation of complexes and suggest that multivalent recognition of CpG-rich RNA targets it for nucleolytic depletion.

The proteasome is a complex protease critical for protein quality control and cell regulation, and its dysfunction is associated with cancer and other diseases. However, the mechanisms that control proteasome activity  in normal and malignant cells remain unclear. Here we report that TRIM11 enhances degradation of aberrant and normal regulatory proteins, and augments overall rate of proteolysis. Mechanistically, TRIM11 binds to both the proteasome and USP14, a deubiquitinase that prematurely removes ubiquitins from proteasome-bound substrates and also noncatalytically inhibits the proteasome, and precludes their association, thereby increasing proteasome activity. TRIM11 promotes cell survival and is upregulated upon heat shock. Moreover, TRIM11 is required for tumor growth, and increased expression of TRIM11 correlates with poor clinical survival. These findings identify TRIM11 as an important activator of the proteasome, define a pathway that adjusts proteasome activity, and reveal a mechanism by which tumor cells acquire higher degradative power to support oncogenic growth. The proteasome-bound ubiquitinase USP14 plays an important role in determining proteasome activity and substrate specificity. Here the authors show that TRIM11, a member of the mammalian tripartite motif family, regulates USP14 and is an important activator of the proteasome.

The hallmark of Salmonella Typhimurium infection is an acute intestinal inflammatory response, which is mediated through the action of secreted bacterial effector proteins. The pro-inflammatory Salmonella effector SopA is a HECT-like E3 ligase, which was previously proposed to activate host RING ligases TRIM56 and TRIM65. Here we elucidate an inhibitory mechanism of TRIM56 and TRIM65 targeting by SopA. We present the crystal structure of SopA in complex with the RING domain of human TRIM56, revealing the atomic details of their interaction and the basis for SopA selectivity towards TRIM56 and TRIM65. Structure-guided biochemical analysis shows that SopA inhibits TRIM56 E3 ligase activity by occluding the E2-interacting surface of TRIM56. We further demonstrate that SopA ubiquitinates TRIM56 and TRIM65, resulting in their proteasomal degradation during infection. Our results provide the basis for how a bacterial HECT ligase blocks host RING ligases and exemplifies the multivalent power of bacterial effectors during infection. The HECT-like E3 ligase SopA in Salmonella has been suggested to activate host RING ligases TRIM56 and TRIM65. Here, the authors use mass spectrometry, crystal structures and biochemistry to examine the interactions between these proteins in detail.