Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 ? crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.     

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.     

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

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.     

MEX is a testis-specific E3 ubiquitin ligase that promotes death receptor-induced apoptosis

In the present study, we report the identification and characterization of MEX (MEKK1-related protein X), a protein with homology to MEKK1 that is expressed uniquely in the testis. MEX is comprises four putative zinc-binding domains including an N-terminal SWIM (SWI2/SNF2 and MuDR) domain of unknown function and two RING (really interesting new gene) fingers separated by a ZZ zinc finger domain. Biochemical analyses revealed that MEX is self-ubiquitinated and targeted for degradation through the proteasome pathway. MEX can act as an E3, Ub (ubiquitin) ligase, through the E2, Ub-conjugating enzymes UbcH5a, UbcH5c or UbcH6. A region of MEX that contains the RING fingers and the ZZ zinc finger was required for interaction with UbcH5a and MEX self-association, whereas the SWIM domain was critical for MEX ubiquitination. The expression of MEX promoted apoptosis that was induced through Fas, DR (death receptor) 3 and DR4 signalling, but not that mediated by the BH3 (Bcl-2 homology 3)-only protein BimEL or the chemotherapeutic drug adriamycin. The enhancement of apoptosis by MEX required a functional SWIM domain, suggesting that MEX ubiquitination is critical for the enhancement of apoptosis. These results indicate that MEX acts as an E3 Ub ligase, an activity that is dependent on the SWIM domain and suggest a role for MEX in the regulation of death receptor-induced apoptosis in the testes.     

Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

Ubiquitylation is a universal mechanism for controlling cellular functions. A large family of ubiquitin E3 ligases (E3) mediates Ubiquitin (Ub) modification. To facilitate Ub transfer, RING E3 ligases bind both the substrate and ubiquitin E2 conjugating enzyme (E2) linked to Ub via a thioester bond to form a catalytic complex. The mechanism of Ub transfer catalyzed by RING E3 remains elusive. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations, we characterized this catalytic mechanism in detail. The three-dimensional model of dimeric RING E3 ligase RNF4 RING, E2 ligase UbcH5A, Ub and the substrate SUMO2 shows close contact between the substrate and Ub transfer catalytic center. Deprotonation of the substrate lysine by D117 on UbcH5A occurs with almost no energy barrier as calculated by MD and QM/MM calculations. Then, the side chain of the activated lysine gets close to the thioester bond via a conformation change. The Ub transfer pathway begins with a nucleophilic addition that forms an oxyanion intermediate of a 4.23 kcal/mol energy barrier followed by nucleophilic elimination, resulting in a Ub modified substrate by a 5.65 kcal/mol energy barrier. These results provide insight into the mechanism of RING-catalyzed Ub transfer guiding the discovery of Ub system inhibitors.     

The basis for selective E1-E2 interactions in the ISG15 conjugation system

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.     

Monoubiquitination Is Critical for Ovarian Tumor Domain-containing Ubiquitin Aldehyde Binding Protein 1 (Otub1) to Suppress UbcH5 Enzyme and Stabilize p53 Protein

Ovarian tumor domain-containing ubiquitin (Ub) aldehyde binding protein 1 (Otub1) regulates p53 stability and activity via non-canonical inhibition of the MDM2 cognate Ub-conjugating enzyme (E2) UbcH5. However, it is not clear how this activity of Otub1 is regulated in cells. Here we report that Otub1 is monoubiquitinated by UbcH5 in cells and in vitro, primarily at the lysine 59 and 109 residues. This monoubiquitination, in turn, contributes to the activity of Otub1 to suppress UbcH5. The lysine-free Otub1 mutant (Otub1^K0) fails to be monoubiquitinated and is unable to suppress the Ub-conjugating activity of UbcH5 in vitro and the MDM2-mediated p53 ubiquitination in cells. Consistently, this mutant is unable to stabilize p53, induce apoptosis, and suppress cell proliferation. Overexpression of Otub1^K0 inhibits DNA-damage induced apoptosis. Adding either Lys-59 or Lys-109 back to the Otub1^K0 mutant restores the monoubiquitination of Otub1 and its function to stabilize and activate p53. We further show that UbcH5 preferentially binds to the monoubiquitinated Otub1 via Ub interaction with its backside donor Ub-interacting surface, suggesting that this binding interferes with the self-assembly of Ub-charged UbcH5 (UbcH5∼Ub) conjugates, which is critical for Ub transfer. Thus, our data reveal novel insights into the Otub1 inhibition of E2 wherein monoubiquitination promotes the interaction of Otub1 with UbcH5 and the function to suppress it.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites

The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We show here that the Caenorhabditis elegans BRCA1/BARD1 (CeBCD) complex possesses an E3-Ub ligase responsible for ubiquitylation at DNA damage sites following ionizing radiation (IR). The DNA damage checkpoint promotes the association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), leading to the formation of an active E3-Ub ligase on chromatin following IR. Correspondingly, defects in Ubc5(let-70) or the DNA damage checkpoint genes atl-1 or mre-11 abolish CeBCD-dependent ubiquitylation in vivo. Extending these findings to human cells reveals a requirement for UbcH5c, the MRN complex, gamma-H2AX and a co-dependence for ATM and ATR kinases for BRCA1-dependent ubiquitylation at DNA damage sites. Furthermore, we demonstrate that the DNA damage checkpoint promotes the association between BRCA1 and UbcH5c to form an active E3-Ub ligase on chromatin after IR. These data reveal that BRCA1-dependent ubiquitylation is activated at sites of DNA repair by the checkpoint as part of a conserved DNA damage response.     

Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.     

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.     

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.     

Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.