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The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.

Eukaryotic cell biology depends on cullin–RING E3 ligase (CRL)-catalysed protein ubiquitylation 1 , which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8 2 – 6 . However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin–RING proteins. A cryo-electron microscopy structure provides insights into the activation of cullin–RING E3 ligases by NEDD8 and the consequent catalysis of ubiquitylation reactions.

The BRCA1–BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination 1 – 10 . The BRCA1–BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets 11 – 14 . The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1–BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks 15 , 16 . We further show that RING domains 17 in BRCA1–BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1–BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1–BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1–BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin 18 – 22 . These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer. The authors elucidate the mechanisms for the ubiquitylation specificity and recruitment of the ubiquitin ligase complex BRCA1–BARD1 to damaged DNA within chromatin to facilitate homologous recombination.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins 1 – 3 . For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters 4 – 6 . How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1–Ub(T)–E2 and E2–Ub(T)–E3 HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next. Structural analyses of analogues of stable ubiquitin transthiolation intermediates with E1, E2 and E3 enzymes reveal a population of intermediate states that provide insights into the directional transfer of ubiquitin between E1, E2 and E3.

Protein misfolding is linked to a wide array of human disorders, including Alzheimer’s disease, Parkinson’s disease and type II diabetes 1 , 2 . Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects 3 – 9 , thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin–proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction. Ubiquitin chains linked to cytoplasmic misfolded proteins are different from those linked to nuclear misfolded proteins, each requiring a distinct combination of molecular chaperones and ubiquitination circuitries.

During protein degradation by the ubiquitin–proteasome pathway, latent 26S proteasomes in the cytosol must assume an active form. Proteasomes are activated when ubiquitylated substrates bind to them and interact with the proteasome-bound deubiquitylase Usp14/Ubp6. The resulting increase in the proteasome’s degradative activity was recently shown to be mediated by Usp14’s ubiquitin-like (Ubl) domain, which, by itself, can trigger proteasome activation. Many other proteins with diverse cellular functions also contain Ubl domains and can associate with 26S proteasomes. We therefore tested if various Ubl-containing proteins that have important roles in protein homeostasis or disease also activate 26S proteasomes. All seven Ubl-containing proteins tested—the shuttling factors Rad23A, Rad23B, and Ddi2; the deubiquitylase Usp7, the ubiquitin ligase Parkin, the cochaperone Bag6, and the protein phosphatase UBLCP1—stimulated peptide hydrolysis two- to fivefold. Rather than enhancing already active proteasomes, Rad23B and its Ubl domain activated previously latent 26S particles. Also, Ubl-containing proteins (if present with an unfolded protein) increased proteasomal adenosine 5′-triphosphate (ATP) hydrolysis, the step which commits substrates to degradation. Surprisingly, some of these proteins also could stimulate peptide hydrolysis even when their Ubl domains were deleted. However, their Ubl domains were required for the increased ATPase activity. Thus, upon binding to proteasomes, Ubl-containing proteins not only deliver substrates (e.g., the shuttling factors) or provide additional enzymatic activities (e.g., Parkin) to proteasomes, but also increase their capacity for proteolysis.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation 1 . In yeast, Ubr1—a single-subunit E3 ligase—is responsible for the Arg/N-degron pathway 2 . How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1. Structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides reveal a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, providing mechanistic insights into the initiation and elongation steps of ubiquitination catalysed by Ubr1.

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes , herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector. During microbial infection, proteins are modified by the ubiquitin-like protein ISG15. Here, the authors uncover RNF213 as a sensor for ISGylated proteins on the surface of lipid droplets, showing that RNF213 has antiviral properties but also directly targets intracellular bacteria in infected cells.

Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification 1 – 3 . The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome 4 , counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1) 5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6 – 9 ). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3–H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A–H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1. The cryo-electron microscopy structure of the Polycomb repressive deubiquitinase (PR-DUB) in complex with the H2AK119ub1 nucleosome provides insight into how the substrate specificity of PR-DUB is achieved.