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1.  Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism 
Structure (London, England : 1993)  2013;21(6):1030-1041.
A distinctive mechanism for ubiquitin (Ub) ligation has recently been proposed for the RING1-IBR-RING2 (RBR) family of E3s: an N-terminal RING1 domain recruits a thioester-linked intermediate complex between Ub and the E2 UbcH7, and a structurally unique C-terminal RING2 domain displays a catalytic cysteine required for Ub ligation. To obtain insights into RBR E3s, we determined the crystal structure of the Human Homolog of Ariadne (HHARI), which reveals the individual RING1, IBR, and RING2 domains embedded in superdomains involving sequences specific to the Ariadne RBR subfamily. The central IBR is flanked on one side by RING1, which is exposed and binds UbcH7. On the other side, a C-terminal autoinhibitory “Ariadne domain” masks the RING2 active site. Insights into RBR E3 mechanisms are provided by structure-based mutations that indicate distinct steps of relief from autoinhibition, Ub transfer from E2 to HHARI, and ligation from the HHARI cysteine to a terminal acceptor.
PMCID: PMC3747818  PMID: 23707686
2.  TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes 
The EMBO Journal  2013;32(21):2848-2860.
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7∼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes
Ubiquitin ligases of the distinct Cullin-RING ligase (CRL) and RING-between-RING (RBR) families physically and functionally interact, suggesting how RBR ligase auto-inhibition may be relieved in Ariadne-subfamily members.
PMCID: PMC3817463  PMID: 24076655
auto-inhibition; Cullin-RING ligases; HHARI; RBR E3 ubiquitin ligases; TRIAD1
3.  Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase 
PLoS ONE  2013;8(8):e74047.
The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn2+-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.
PMCID: PMC3772753  PMID: 24058416
4.  Sequence Determinants of E2-E6AP Binding Affinity and Specificity 
Journal of molecular biology  2007;369(2):419-428.
The conjugation of ubiquitin to substrates requires a series of enzymatic reactions consisting of an activating enzyme (E1), conjugating enzymes (E2) and ligases (E3). Tagging the appropriate substrate with ubiquitin is achieved by specific E2-E3 and E3-substrate interactions. E6AP, a member of the HECT family of E3s, has been previously shown to bind and function with the E2s UbcH7 and UbcH8. To decipher the sequence determinants of this specificity we have developed a quantitative E2-E3 binding assay based on fluorescence polarization and used this assay to measure the affinity of wild type and mutant E2–E6AP interactions. Alanine scanning of the E6AP–UbcH7 binding interface identified 4 side chains on UbcH7 and 6 side chains on E6AP that contribute more than 1 kcal /mol to the binding free energy. Two of the hot spot residues from UbcH7 (K96 and K100) are conserved in UbcH8 but vary across other E2s. To determine if these are key specificity determining residues, we attempted to induce a tighter association between the E2 UbcH5b and E6AP by mutating the corresponding positions in UbcH5b to lysines. Surprisingly, the mutations had little effect, but rather a mutation at UbcH7 position 4, which is not at a hot spot on the UbcH7–E6AP interface, significantly strengthened UbcH5bs affinity for E6AP. This result indicates that E2-E3 binding specificities are a function of both favorable interactions that promote binding, and unfavorable interactions that prevent binding with unwanted partners.
PMCID: PMC1945100  PMID: 17433363
Ubiquitin; UbcH7; E6AP; HECT; E2-E3 Specificity
5.  The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension 
The EMBO Journal  2012;31(19):3833-3844.
The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension
Like Parkin, the linear ubiquitin chain assembly complex LUBAC functions as a RING/HECT-hybrid ubiquitin ligase, but includes a unique extension that dictates linear ubiquitin linkage specificity.
Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains.
PMCID: PMC3463842  PMID: 22863777
E3 ligase; HHARI; Parkin; RNF31; TRIAD
6.  Structure of BIRC7–E2 ubiquitin conjugate reveals the mechanism of ubiquitin transfer by a RING dimer 
Nature structural & molecular biology  2012;19(9):10.1038/nsmb.2379.
RING ubiquitin ligases (E3s) recruit E2 thioesterified with Ub to facilitate Ub transfer to a target. Certain RING E3s dimerize to form active ligases but structural evidence on how this process promotes Ub transfer is lacking. Several members of the baculovirus inhibitor of apoptosis repeat-containing (BIRC) family of proteins function as dimeric RING E3s in the regulation of cell death. Here we report the structure of the human dimeric RING domain from BIRC7 in complex with the E2 UbcH5B covalently linked to Ub at its active site (UbcH5B~Ub). In addition to the known E2–RING contacts, the structure reveals extensive non-covalent donor Ub interactions with UbcH5B and both subunits of the RING domain dimer. Mutations that disrupt these non-covalent interactions or RING dimerization reduce UbcH5B~Ub binding affinity and ubiquitination activity. Moreover, NMR analyses demonstrate that BIRC7 binding to UbcH5B~Ub induces peak shift perturbations in the donor Ub consistent with the crystallographically-observed BIRC7–Ub interactions. Our results provide structural insights into how dimeric RING E3s recruit E2~Ub and optimize the donor Ub configuration for transfer.
PMCID: PMC3880866  PMID: 22902369
Inhibitor of apoptosis; BIRC7; E3; E2 ubiquitin conjugate; RING dimerization
7.  RING-H2 Protein WSSV249 from White Spot Syndrome Virus Sequesters a Shrimp Ubiquitin-Conjugating Enzyme, PvUbc, for Viral Pathogenesis 
Journal of Virology  2005;79(14):8764-8772.
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.
PMCID: PMC1168725  PMID: 15994770
8.  The Unique N-terminus of the UbcH10 E2 Enzyme Controls the Threshold for APC Activation and Enhances Checkpoint Regulation of the APC 
Molecular cell  2008;31(4):544.
In vitro, the Anaphase Promoting Complex (APC) E3 ligase functions with E2 ubiquitin conjugating enzymes of the E2–C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2–C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2–E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N-terminus increases substrate ubiquitination, the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction-boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N-terminus to UbcH5 restricts ubiquitination activity, but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2–E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.
PMCID: PMC2813190  PMID: 18722180
Mitosis; Emi1; Ubiquitin-Protein Ligases; UbcH10; Anaphase Promoting Complex; Spindle Assembly Checkpoint
9.  Interactions between the quality control ubiquitin ligase CHIP and ubiquitin conjugating enzymes 
Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.
The 2.9 Å crystal structure of the CHIP U-box domain complexed with UbcH5a shows that CHIP binds to UbcH5 and Ubc13 through similar specificity determinants, including a key S-P-A motif on the E2 enzymes. The determinants make different relative contributions to the overall interactions between CHIP and the two E2 enzymes. CHIP undergoes auto-ubiquitination by UbcH5 but not by Ubc13-Uev1a. Instead, CHIP drives the formation of unanchored polyubiquitin by Ubc13-Uev1a. CHIP also interacts productively with the class III E2 enzyme Ube2e2, in which the UbcH5- and Ubc13-binding specificity determinants are highly conserved.
The CHIP:UbcH5a structure emphasizes the importance of specificity determinants located on the long loops and central helix of the CHIP U-box, and on the N-terminal helix and loops L4 and L7 of its cognate E2 enzymes. The S-P-A motif and other specificity determinants define the set of cognate E2 enzymes for CHIP, which likely includes several Class III E2 enzymes. CHIP's interactions with UbcH5, Ube2e2 and Ubc13-Uev1a are consistent with the notion that Ubc13-Uev1a may work sequentially with other E2 enzymes to carry out K63-linked polyubiquitination of CHIP substrates.
PMCID: PMC2396629  PMID: 18485199
10.  RBR E3 ubiquitin ligases: new structures, new insights, new questions 
Biochemical Journal  2014;458(Pt 3):421-437.
The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.
PMCID: PMC3940038  PMID: 24576094
catalysis; structure; ubiquitination; ubiquitin ligase; ANKIB1, ankyrin repeat- and IBR domain-containing 1; BRcat, benign-catalytic; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Cdk5, cyclin-dependent kinase 5; cIAP2, cellular inhibitor of apoptosis 2; CK1, casein kinase 1; CPH, Cul7, Parc and HERC2 proteins; CRL, Cul-RING-ligase; Cul, cullin; Eps15, epidermal growth factor receptor pathway substrate 15; FANCL, Fanconi anaemia, complementation group L; HDAC, histone deacetylase; HECT, homologous with E6-associated protein C-terminus; HOIL-1, haem-oxidized IRP2 ubiquitin ligase 1; HOIP, HOIL-1-interacting protein; IBR, InBetweenRING; LUBAC, linear ubiquitin chain assembly complex; MDM2, murine double minute 2; MIRO, mitochondrial Rho GTPase; NEDD, neural-precursor-cell-expressed developmentally down-regulated; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; NZF, Npl4 ZNF; Parc, parkin-like cytoplasmic p53-binding protein; PINK1, PTEN-induced putative kinase 1; PKC, protein kinase C; RanBP2, RAN-binding protein 2; RBR, RING-BetweenRING-RING/RING1-BRcat-Rcat; Rcat, required-for-catalysis; RNF, RING finger protein; RWD, RING finger and WD repeat-containing; SH3, Src homology 3; SHARPIN, SHANK-associated RH domain interactor; SILAC, stable isotope labelling by amino acids in cell culture; SUMO, small ubiquitin-related modifier; TOMM70A, translocase of outer mitochondrial membrane 70 homologue A; TRAF6, tumour-necrosis-factor-receptor-associated factor 6; TRIAD, two RING fingers and a DRIL (double RING finger linked); UBA, ubiquitin-associated; UBE2L, ubiquitin-conjugating enzyme E2L; UIM, ubiquitin-interacting motif; Ubl, ubiquitin-like; ZNF, zinc finger
11.  Insights into ubiquitin transfer cascades from a structure of a UbcH5B~Ubiquitin-HECTNEDD4L complex 
Molecular cell  2009;36(6):1095-1102.
In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2~Ub covalent complex, and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor Cys in the HECT domain C-lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently-linked Ub at its active site (UbcH5B~Ub). Noncovalent interactions between UbcH5B and the HECT N-lobe and between Ub and the HECT domain C-lobe lead to an overall compact structure, with the Ub C-terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.
PMCID: PMC2859195  PMID: 20064473
Ubiquitin; HECT; E3; Ubiquitin ligase; UbcH5B; NEDD4L; NEDD4-2
12.  The Stability of Herpes Simplex Virus 1 ICP0 Early after Infection Is Defined by the RING Finger and the UL13 Protein Kinase 
Journal of Virology  2014;88(10):5437-5443.
Herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a multifunctional protein that plays a key role in overcoming numerous facets of host innate immunity. A key function of ICP0 that requires an intact RING finger domain is that of an ubiquitin E3 ligase: ICP0 interacts with at least three ubiquitin-conjugating enzymes of which one, UbcH5a, is required for degradation of PML and SP100. A preceding report showed that ICP0 is highly unstable at very early times after infection but becomes stable at later times. We report here that (i) the degradation of ICP0 is not infected cell specific, (ii) the degradation does not require the interaction of ICP0 with either UbcH5a, UbcH6, or UbcH9, (iii) ICP0 is degraded both early and late in cells infected with a mutant lacking the UL13 protein kinase, (iv) ICP0 encoded by wild-type virus or the ΔUL13 mutant is stable in cells transfected with a plasmid encoding UL13 before infection, (v) ICP0 carrying mutations in the RING finger domain is stable both early and late in infection, and, finally, (vi) in cells infected with both wild type and RING finger mutant only the wild-type ICP0 is rapidly degraded at early times. The results suggest that the stability of ICP0 is mediated by the UL13 protein kinase and that the target of proteolysis is a site at or near the RING domain of ICP0.
IMPORTANCE ICP0, a major regulatory protein of HSV-1, turns over rapidly early in infection but becomes stable at late times. We report that stabilization requires the presence of UL13 protein kinase and that an ICP0 with mutations in RING finger is stable. In mixed infections mutant ICP0 is stable, whereas the wild-type ICP0 is degraded. Our findings suggest that the lifestyle of HSV-1 requires an ICP0 that turns over rapidly if late proteins are absent.
PMCID: PMC4019132  PMID: 24574411
13.  Structure of the human Parkin ligase domain in an autoinhibited state 
The EMBO Journal  2013;32(15):2099-2112.
Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING-in-between-RING(IBR)-RING (RBR) family, which, in an HECT-like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn-binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50-Å distance blocks the conserved E2∼Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra-domain interfaces activates Parkin, and enables Ub-based suicide probes to modify Cys431. The structure further reveals a putative phospho-peptide docking site in the UPD, and explains many PD-causing mutations.
Structure of the human Parkin ligase domain in an autoinhibited state
The complete structural view of a RING-IBR-RING (RBR) ubiquitin ligase domain reveals an unexpected catalytic triad and explains the effects of various Parkin mutations underlying Parkinson's disease.
PMCID: PMC3730226  PMID: 23727886
E3 ligase; neurodegenerative disease; Parkin; ubiquitin; X-ray crystallography
14.  Genome-Wide Identification and Characterization of RBR Ubiquitin Ligase Genes in Soybean 
PLoS ONE  2014;9(1):e87282.
RBR (RING1-IBR-RING2) proteins play an important role in protein ubiquitination and are involved in many cellular processes. Recent studies showed plant RBR genes were induced by abiotic and biotic stresses. However, detailed studies on RBR genes in the important oil crop, soybean (Glycine max (L.) Merr.), is still lacking. Here we performed a genome-wide search and identified 24 RBR domain-containing genes from the soybean genome sequence and cloned 11 of them. Most soybean RBR proteins contain a highly conserved RBR supra-domain. Phylogenetic analyses indicated all 24 soybean RBR proteins are most related to the RBR proteins from Phaseolus vulgaris, and could be classified into seven groups including Ariadne A, Ariadne B, ARA54, Plant IIA, Plant IIB, Plant IIC, and Helicase. Tandem duplication and block duplication were found among the Ariadne B and Plant IIC group of soybean RBR genes. Despite the conserved RBR supra-domain, there are extensive variations in the additional protein motifs and exon-intron structures between different groups, which indicate they might have diverse functions. Most soybean RBR proteins are predicted to localize in nucleus, and four of them were experimentally confirmed by GFP fusion proteins. Soybean RBR genes are broadly expressed in many tissue types with a little more abundant in the roots and flowers than leaves, stems, and seeds. The expression of GmRTRTP3 (Plant IIB) and GmRTRTP5 (Plant IIC) are induced by NaCl treatment, which suggests these RBR genes might be involved in soybean response to abiotic stresses.
PMCID: PMC3904995  PMID: 24489889
15.  Overexpression of the E2 ubiquitin–conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation 
The Journal of Cell Biology  2010;188(1):83-100.
An overabundance of UbcH10 disrupts mitotic checkpoint signaling as a result of a degradation of cyclin B, increasing spontaneous and carcinogen-induced tumor formation in transgenic mice.
The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin–conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.
PMCID: PMC2812857  PMID: 20065091
16.  Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations 
PLoS ONE  2014;9(7):e101663.
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.
PMCID: PMC4086935  PMID: 25003393
17.  Structure of human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7) 
The crystal structure of human UBE2G2/UBC7 was solved at 2.56 Å resolution. The superimposition of UBE2G2 on UbcH7 in a c-Cbl–UbcH7–ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way as UbcH7.
The human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7) is involved in protein degradation, including a process known as endoplasmic reticulum-associated degradation (ERAD). The crystal structure of human UBE2G2/UBC7 was solved at 2.56 Å resolution. The UBE2G2 structure comprises a single domain consisting of an antiparallel β-sheet with four strands, five α-­helices and two 310-helices. Structural comparison of human UBE2G2 with yeast Ubc7 indicated that the overall structures are similar except for the long loop region and the C-terminal helix. Superimposition of UBE2G2 on UbcH7 in a c-Cbl–UbcH7–ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way to UbcH7. In addition, the extra loop region of UBE2G2 may interact with the RING domain or its neighbouring region and may be involved in the binding specificity and stability.
PMCID: PMC2222581  PMID: 16582478
ubiquitin-conjugating enzyme; UBE2G2/UBC7; ubiquitin-dependent protein degradation; endoplasmic reticulum-associated degradation; Parkin
18.  LUBAC synthesizes linear ubiquitin chains via a thioester intermediate 
EMBO Reports  2012;13(9):840-846.
LUBAC synthesizes linear ubiquitin chains via a thioester intermediate
The N-terminus of the LUBAC catalytic subunit is shown to be autoinhibitory and counteracted by the other subunits of the complex. Linear ubiquitination proceeds through a thioesther intermediate, indicative of a RING/HECT hybrid mechanism.
The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP—the catalytic subunit of LUBAC—is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.
PMCID: PMC3432797  PMID: 22791023
E3 ligase; mechanism; thioester; ubiquitination
19.  Tumor suppressor candidate TSSC5 is regulated by UbcH6and a novel ubiquitin ligase RING105 
Oncogene  2006;25(9):1330-1339.
The region of human chromosome 11p15.5 is linked with Beckwith–Wiedemann syndrome that is associated with susceptibility to Wilms’ tumor, rhabdomyosarcoma and hepatoblastoma. TSSC5 (tumor-suppressing subchromosomal transferable fragment cDNA; also known as ORCTL2/IMPT1/BWR1A/SLC22A1L) is located in the region. The expression of TSSC5 and other genes in the region is regulated through paternal imprinting. Mutations and/or reduced expression of TSSC5 have been found in certain tumors. TSSC5 encodes an efflux transporter-like protein with 10 transmembrane domains, whose regulation may affect drug sensitivity, cellular metabolism and growth. Here, we present evidences indicating that RING105, a novel conserved RING-finger protein with a PA (protease-associated) domain and a PEST sequence, is a ubiquitin ligase for TSSC5 that can function in concert with the ubiquitin-conjugating enzyme UbcH6. The polyubiquitin target site on TSSC5 was mapped to a region in the 6th hydrophilic loop. Ectopic expression of RING105 in HeLa cells caused an accumulation of cells during G1 that was not observed with the expression of a form of RING105 in which a residue within the RING finger was mutated to inactivate its ligase activity. UbcH6-RING105 may define a novel ubiquitin–proteasome pathway that targets TSSC5 in mammalian cells.
PMCID: PMC2713668  PMID: 16314844
TSSC5; protease associated; RING finger; ubiquitin ligase; UbcH6; 11p15.5
20.  Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3 
eLife  2013;2:e00828.
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.
eLife digest
Ubiquitin is a small protein that can be covalently linked to other, ‘target’, proteins in a cell to influence their behavior. Ubiquitin can be linked to its targets either as single copies or as polyubiquitin chains in which several ubiquitin molecules are bound end-on-end to each other, with one end of the chain attached to the target protein. A multi-step cascade involving enzymes known as E1, E2, and E3 adds ubiquitin to its targets. These enzymes function in a manner like runners in a relay, with ubiquitin a baton that is passed from E1 to E2 to E3 to the target.
The E3 enzyme is a ligase that catalyzes the formation of a new chemical bond between a ubiquitin and its target. There are approximately 600 different E3 enzymes in human cells that regulate a wide variety of target proteins. A major class of E3 enzymes, called HECT E3s, attaches ubiquitin to its targets in a unique two-step mechanism: the E2 enzymes covalently link a ubiquitin to a HECT E3 to form a complex that subsequently transfers the ubiquitin to its target protein. The ubiquitin is typically added to a particular amino acid, lysine, on the target protein, but the details of how HECT E3s execute this transfer are not well understood. To address this issue, Kamadurai et al. investigate how Rsp5, a HECT E3 ligase in yeast, attaches ubiquitin to a target protein called Sna3.
All HECT E3s have a domain—the HECT domain—that catalyzes the transfer of ubiquitin to its target protein. This domain consists of two sub-structures: the C-lobe, which can receive ubiquitin from E2 and then itself become linked to ubiquitin, and the N-lobe. These lobes were previously thought to adopt various orientations relative to each other to deliver ubiquitin to sites on different target proteins (including to multiple lysines on a single target protein). Unexpectedly, Kamadurai et al. find that in order to transfer the ubiquitin to Sna3, Rsp5 adopts a discrete HECT domain architecture that creates an active site in which parts of the C-lobe and the N-lobe, which are normally separated, are brought together with a ubiquitin molecule. This architecture also provides a mechanism that dictates which substrate lysines can be ubiquitinated based on how accessible they are to this active site.
The same regions of Rsp5 transfer ubiquitin to targets other than Sna3, suggesting that a uniform mechanism—which Kamadurai et al. show is conserved in two related human HECT E3 ligases—might transfer ubiquitin to all its targets. These studies therefore represent a significant step toward understanding how a major class of E3 enzymes modulates the functions of their targets.
PMCID: PMC3738095  PMID: 23936628
ubiquitin; HECT; E3 ligase; E2 conjugating enzyme; NEDD4; Rsp5; S. cerevisiae
21.  Mechanism of ubiquitylation by dimeric RING ligase RNF4 
Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the “Ile44 hydrophobic patch” of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.
PMCID: PMC3326525  PMID: 21857666
22.  Structure of the HECT C-lobe of the UBR5 E3 ubiquitin ligase 
The structure of the HECT C-lobe of UBR5 shows a unique tertiary conformation. The sub-domain is also able to form a thioester bond with ubiquitin and may thus confer E2 binding specificity to UBR5.
UBR5 ubiquitin ligase (also known as EDD, Rat100 or hHYD) is a member of the E3 protein family of HECT (homologous to E6-AP C-terminus) ligases as it contains a C-terminal HECT domain. In ubiquitination cascades involving E3s of the HECT class, ubiquitin is transferred from an associated E2 ubiquitin-conjugating enzyme to the acceptor cysteine of the HECT domain, which consists of structurally distinct N- and C-lobes connected by a flexible linker. Here, the high-resolution crystal structure of the C-lobe of the HECT domain of human UBR5 is presented. The structure reveals important features that are unique compared with other HECT domains. In particular, a distinct four-residue insert in the second helix elongates this helix, resulting in a strikingly different orientation of the preceding loop. This protruding loop is likely to contribute to specificity towards the E2 ubiquitin-conjugating enzyme UBCH4, which is an important functional partner of UBR5. Ubiquitination assays showed that the C-lobe of UBR5 is able to form a thioester-linked E3–ubiquitin complex, although it does not physically interact with UBCH4 in NMR experiments. This study contributes to a better understanding of UBR5 ubiquitination activity.
PMCID: PMC3497971  PMID: 23027739
UBR5; HECT domains; E3 ubiquitin ligases; C-lobe
23.  Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases 
Nature Communications  2013;4:1982.
Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.
The Parkinson’s disease-associated protein Parkin regulates the fate of damaged mitochondria by ubiquitinating mitochondrial substrates. Riley et al. present the crystal structure of the Parkin-R0RBR domain, providing new insight into the catalytic mechanism of the enzyme.
PMCID: PMC3709503  PMID: 23770887
24.  Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP 
Nature  2013;503(7476):422-426.
Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis1-5. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multi-subunit E3 ligase6. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin7. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC.
PMCID: PMC3838313  PMID: 24141947
E3 ubiquitin ligase; linear ubiquitin chains; structure; mechanism; ubiquitination
25.  Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7 
Cell Division  2009;4:17.
Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increases in gene products dictated by changes in transcription. Of the dozens of ubiquitin conjugating enzymes, or E2s, functions in control of the cell cycle have been defined for only UbcH10 and Ubc3/Cdc34. Each of these E2s works primarily with one ubiquitin ligase or E3. Here we show that another E2, UbcH7 is a regulator of S phase of the cell cycle. Over-expression of UbcH7 delays entry into S phase whereas depletion of UbcH7 increases the length of S phase and decreases cell proliferation. Additionally, the level of the checkpoint kinase Chk1 increases upon UbcH7 depletion while the level of phosphorylated PTEN decreases. Taken together, these data indicate that the length of S phase is controlled in part by UbcH7 through a PTEN/Akt/Chk1 pathway. Potential mechanisms by which UbcH7 controls Chk1 levels both directly and indirectly, as well as the length of S phase are discussed and additional functions for UbcH7 are reviewed.
PMCID: PMC2734563  PMID: 19664228

Results 1-25 (847259)