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Cellular adaptation to proteotoxic stress at the endoplasmic reticulum (ER) depends on Lys48-linked polyubiquitination by ER-associated ubiquitin ligases (E3s) and subsequent elimination of ubiquitinated retrotranslocation products by the proteasome. The ER-associated E3 gp78 ubiquitinates misfolded proteins by transferring preformed Lys48-linked ubiquitin chains from the cognate E2 Ube2g2 to substrates. Here we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER-associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self-association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48-linked ubiquitin chains.
Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.
Polyubiquitination is important in controlling NF-κB signaling. Excessive NF-κB activity has been linked to inflammatory disorders and autoimmune diseases, while ABIN1 could attenuate NF-κB activation to maintain immune homeostasis by utilizing UBAN to recognize linear (M1)-linked polyubiquitinated NF-κB activation mediators, including NEMO, IRAK1 and RIP1. PolyUb-mediated UBAN recruitment remains undetermined, since the recognition studies focused mostly on di-ubiquitin (diUb). Here we report three crystal structures of human ABIN1 UBAN (hABIN1UBAN) in complex with M1-linked diUb, triUb, and tetraUb, respectively. Notably, the hABIN1UBAN:diUb structure reveals that a diUb randomly binds one of the Ub-binding sites of the hABIN1UBAN dimer and leaves the other site vacant. Together with the ITC and gel-filtration analyses, we found that M1-triUb and M1-tetraUb adopt two unique conformations, instead of an elongated one, and they preferentially use the N-terminal two-Ub unit to bind the primary Ub-binding site of a hABIN1UBAN dimer and the C-terminal two-Ub unit to bind the secondary Ub-binding site of another hABIN1UBAN dimer. Especially, our results suggest that two ABIN1UBAN dimers cooperatively bind two UBAN-binding units of a tetraUb or vice versa. Since the UBAN family members share a conserved diUb-binding mode, our results suggest that M1-polyUb modification allows multiple copies of the two-tandem Ub unit to simultaneously coordinate multiple and/or different binding partners to increase their local concentrations and to facilitate the formation of a large signaling complex. Our study provides a structural-functional glimpse of M1-polyUb as a multiple-molecule binding platform to exert its intrinsic structural plasticity in mediating cellular signaling.
The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.
Ubiquitin mediated signaling contributes critically to host cell defenses during pathogen infection. Many pathogens manipulate the ubiquitin system to evade these defenses. Here we characterize a likely effector protein bearing a deubiquitylase (DUB) domain from the obligate intracellular bacterium Orientia tsutsugamushi, the causative agent of scrub typhus. The Ulp1-like DUB prefers ubiquitin substrates over ubiquitin-like proteins and efficiently cleaves polyubiquitin chains of three or more ubiquitins. The co-crystal structure of the DUB (OtDUB) domain with ubiquitin revealed three bound ubiquitins: one engages the S1 site, the second binds an S2 site contributing to chain specificity and the third binds a unique ubiquitin-binding domain (UBD). The UBD modulates OtDUB activity, undergoes a pronounced structural transition upon binding ubiquitin, and binds monoubiquitin with an unprecedented ~5 nM dissociation constant. The characterization and high-resolution structure determination of this enzyme should aid in its development as a drug target to counter Orientia infections.
Post-translational substrate modification with ubiquitin is essential for eukaryotic cellular signaling. Polymeric ubiquitin chains are assembled with specific architectures, which convey distinct signaling outcomes depending on the linkages involved. Recently, branched K11/K48-linked polyubiquitins were shown to enhance proteasomal degradation during mitosis. To better understand the underlying structural mechanisms, we determined the crystal and NMR structures of branched K11/K48-linked tri-ubiquitin and discovered a previously unobserved interdomain interface between the distal ubiquitins. Small-angle neutron scattering and site-directed mutagenesis corroborated the presence of this interface, which we hypothesized to be influential in the physiological role of branched K11/K48-linked chains. Yet, experiments probing polyubiquitin interactions-deubiquitination assays, binding to proteasomal shuttle hHR23A-showed negligible differences between branched K11/K48-linked tri-ubiquitin and related di-ubiquitins. However, significantly stronger binding affinity for branched K11/K48-linked tri-ubiquitin was observed with proteasomal subunit Rpn1, thereby suggesting a functional impact of this interdomain interface and pinpointing the mechanistic site of enhanced degradation.
Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.
Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress.
Toxic protein aggregates are key features of progressive neurodegenerative diseases. In addition to "seed" proteins diagnostic for each neuropathy (e.g., Aβ1-42 and tau in Alzheimer's disease), aggregates contain numerous other proteins, many of which are common to aggregates from diverse diseases. We reported that CRAM-1, discovered in insoluble aggregates of C. elegans expressing Q40::YFP, blocks proteasomal degradation of ubiquitinated proteins and thus promotes aggregation. We now show that CRAM-1 contains three α-helical segments forming a UBA-like domain, structurally similar to those of mammalian adaptor proteins (e.g. RAD23, SQSTM1/p62) that shuttle ubiquitinated cargos to proteasomes or autophagosomes for degradation. Molecular modeling indicates that CRAM-1, through this UBA-like domain, can form tight complexes with mono- and di-ubiquitin and may thus prevent tagged proteins from interacting with adaptor/shuttle proteins required for degradation. A human ortholog of CRAM-1, SERF2 (also largely disordered), promotes aggregation in SH-SY5Y-APPSw human neuroblastoma cells, since SERF2 knockdown protects these cells from amyloid formation. Atomistic molecular-dynamic simulations predict spontaneous unfolding of SERF2, and computational large-scale protein-protein interactions predict its stable binding to ubiquitins. SERF2 is also predicted to bind to most proteins screened at random, although with lower average stability than to ubiquitins, suggesting roles in aggregation initiation and/or progression.
Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent.
The ability of ubiquitin to function in a wide range of cellular processes is ascribed to its capacity to cause a diverse spectrum of modifications. While a target protein can be modified with monoubiquitin, it can also be modified with ubiquitin chains. The latter include seven types of homotypic chains as well as mixed ubiquitin chains. In a mixed chain, not all the isopeptide bonds are restricted to a specific lysine of ubiquitin, resulting in a chain possessing more than one type of linkage. While structural characterization of homotypic chains has been well elucidated, less is known about mixed chains. Here we present the crystal structure of a mixed tri-ubiquitin chain at 3.1-Å resolution. In the structure, the proximal ubiquitin is connected to the middle ubiquitin via K48 and these two ubiquitins adopt a compact structure as observed in K48 di-ubiquitin. The middle ubiquitin links to the distal ubiquitin via its K63 and these ubiquitins adopt two conformations, suggesting a flexible structure. Using small-angle X-ray scattering, we unexpectedly found differences between the conformational ensembles of the above tri-ubiquitin chains and chains possessing the same linkages but in the reverse order. In addition, cleavage of the K48 linkage by DUB is faster if this linkage is at the distal end. Taken together, our results suggest that in mixed chains, not only the type of the linkages but also their sequence determine the structural and functional properties of the chain.
A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.
While the signaling properties of ubiquitin depend on the topology of polyubiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyubiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyubiquitin signals.
Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.
Polyubiquitin chains are flexible multidomain proteins, whose conformational dynamics enable them to regulate multiple biological pathways. Their dynamic is determined by the linkage between ubiquitins and by the number of ubiquitin units. Characterizing polyubiquitin behavior as a function of their length is hampered because of increasing system size and conformational variability. Here, we introduce a new approach to efficiently integrating small-angle x-ray scattering with simulations allowing us to accurately characterize the dynamics of linear di-, tri-, and tetraubiquitin in the free state as well as of diubiquitin in complex with NEMO, a central regulator in the NF-κB pathway. Our results show that the behavior of the diubiquitin subunits is independent of the presence of additional ubiquitin modules and that the dynamics of polyubiquitins with different lengths follow a simple model. Together with experimental data from multiple biophysical techniques, we then rationalize the 2:1 NEMO:polyubiquitin binding.
Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.
Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications.
The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposi's sarcoma-associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex (MHC) class I molecules. Here, we show that K5 targets the membrane proximal lysine of MHC I for conjugation with mixed linkage polyubiquitin chains. Quantitative mass spectrometry revealed an increase in lysine-11, as well as lysine-63, linked polyubiquitin chains on MHC I in K5-expressing cells. Using a combination of mutant ubiquitins and MHC I molecules expressing a single cytosolic lysine residue, we confirm a functional role for lysines-11 and -63 in K5-mediated MHC I endocytosis. We show that lysine-11 linkages are important for receptor endocytosis, and that complex mixed linkage polyubiquitin chains are generated in vivo.
Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.
Rpn13/Adrm1 is recruited to the proteasome by PSMD1/Rpn2, where it serves as a substrate receptor that binds preferentially to K48-linked ubiquitin chains, an established signal for protein proteolysis. Here, we use NMR to solve the structure of hRpn13 Pru:hRpn2 (940-953):K48-diubiquitin. Surprisingly, hRpn2-bound hRpn13 selects a dynamic, extended conformation of K48-diubiquitin that is unique from previously determined structures. NMR experiments on free K48-diubiquitin demonstrate the presence of the reported "closed" conformation observed by crystallography, but also this more extended state, in which the hRpn13-binding surface is exposed. This extended K48-diubiquitin conformation is defined by interactions between L73 from G76-linked (distal) ubiquitin and a Y59-centered surface of K48-linked (proximal) ubiquitin. Furthermore, hRpn13 exchanges between the two ubiquitins within 100 ms, although prefers the proximal ubiquitin due to interactions with the K48 linker region. Altogether, these data lead to a revised model of how ubiquitinated substrates interact with the proteasome.
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