K27‐linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly‐ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell‐based ubiquitin replacement strategy to enable selective and conditional abrogation of K27‐linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27‐linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97‐proteasome pathway model substrate (Ub(G76V)‐GFP) is directly modified by K27‐linked ubiquitylation, and that disabling the formation of K27‐linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage‐specific binder UCHL3 impedes Ub(G76V)‐GFP turnover at the level of p97 function. Our findings suggest a critical role of K27‐linked ubiquitylation in supporting cell fitness by facilitating p97‐dependent processing of ubiquitylated nuclear proteins.     

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors.     

The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein–protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain–domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.     

The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show that both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.     

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N‐terminal ubiquitin‐like (UBL) and C‐terminal ubiquitin‐associated (UBA) domains, respectively. Between these two folded domains are low‐complexity STI1‐I and STI1‐II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1‐II region enables UBQLN2 to undergo liquid–liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well‐studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1‐I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.     

Ubiquitin acetylation inhibits polyubiquitin chain elongation

Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B—which we identify as an endogenous substrate of acetylated ubiquitin—and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono- and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology.     

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.     

Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation

Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-kappaB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2-Ubc13-ubiquitin (UEV-E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2-Ubc13-Ub complex to the acceptor-binding site of Mms2-Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.     

Modulation of K11-linkage formation by variable loop residues within UbcH5A

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.     

A heterotypic assembly mechanism regulates CHIP E3 ligase activity

CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation.     

Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair

Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.     

Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain

TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.     

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic‐type serine‐threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG–host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin‐activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor‐associated factor 2 (TRAF2) and TGF‐β‐activated kinase 1 (TAK1), thereby inhibiting the activation of NF‐κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin‐conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82‐Ub), rather than the usual C86‐Ub thiol‐ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.     

Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.     

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron‐enriched genes

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron‐specific ChIP‐seq and RNA‐seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron‐specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B‐dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.     

E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages

An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.     

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys(48)-linked chains target proteins for proteasomal degradation, and Lys(63)-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13-Uev1a supports the formation of free Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.     

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.     

Structural basis for feedforward control in the PINK1/Parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin‐like (Ubl) domain of parkin. Here, we observed that phospho‐ubiquitin can bind to two distinct sites on parkin, a high‐affinity site on RING1 that controls parkin localization and a low‐affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho‐ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra‐phospho‐ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1‐parkin pathway and likely represents an intermediate step in its evolutionary development.     

Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2

Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating enzyme variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.