Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.     

A Structural Element within the HUWE1 HECT Domain Modulates Self-ubiquitination and Substrate Ubiquitination Activities

E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.     

β-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe ~ Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the β-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub.     

WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

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.     

Structure of the human Parkin ligase domain in an autoinhibited state

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.     

Interaction Between Syntaxin 8 and HECTd3, a HECT Domain Ligase

Ubiquitination of proteins and their degradation within the proteasome has emerged as the major proteolytic mechanism used by mammalian cells to regulate cytosolic and nuclear protein levels. Substrate ubiquitylation is mediated by ubiquitin (Ub) ligases, also called E3 Ub ligases. HECT-E3 Ub ligases are characterized by the presence of a C-terminal HECT domain that contains the active site for Ub transfer onto substrates. Among the many E3 Ub ligases, the family homologous to E6-Ap C-terminus (HECT) E3 Ub ligases, which includes the yeast protein Rsp5p and the mammalian homolog NEDD4, AIP4/Itch, and Smurf, has been shown to ubiquitylate membrane proteins and, in some instances, to induce their degradation. In this report, we have identified Syntaxin 8 as a binding protein to a novel HECT domain protein, HECT domain containing 3 (HECTd3), by yeast two-hybrid screen. Besides HECT domain, HECTd3 contains an anaphase-promoting complex, subunit 10 (APC10) domain. Our co-immunoprecipitation experiments show that Syntaxin 8 directly interacts with HECTd3 and that the overexpression of HECTd3 promotes the ubiquitination of Syntaxin 8. Immunofluorescence results show that Syntaxin 8 and HECTd3 have similar subcellular localization.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

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.     

Ubiquitin‐Activated Interaction Traps (UBAITs) identify E3 ligase binding partners

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ub iquitin‐A ctivated I nteraction T raps) are E3‐ubiquitin fusion proteins and, in an E1‐ and E2‐dependent manner, the C‐terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co‐purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester‐linked lariat intermediate or through an E2 thioester intermediate, and both WT and active‐site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double‐strand break repair. Using the RNF168 UBAIT, we identify H2AZ—a histone protein involved in DNA repair—as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.     

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.     

Allosteric auto‐inhibition and activation of the Nedd4 family E3 ligase Itch

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto‐inhibition. However, the molecular mechanism underlying Nedd4 E3 auto‐inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2‐E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1‐mediated phosphorylation relieves the auto‐inhibition of Itch in a WW2‐dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.     

The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only monoubiquitin transfer by Ubc4

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5- and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2～Ub complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.     

Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.     

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.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.     

Loop 7 of E2 enzymes: an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination cascade

The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3, and influencing the ultimate fate of the substrates. Several E2s are characterized by an extended acidic insertion in loop 7 (L7), which if mutated is known to impair the proper E2-related functions. In the present contribution, we show that acidic loop is a conserved ancestral motif in E2s, relying on the presence of alternate hydrophobic and acidic residues. Moreover, the dynamic properties of a subset of family 3 E2s, as well as their binary and ternary complexes with Ub and the cognate E3, have been investigated. Here we provide a model of L7 role in the different steps of the ubiquitination cascade of family 3 E2s. The L7 hydrophobic residues turned out to be the main determinant for the stabilization of the E2 inactive conformations by a tight network of interactions in the catalytic cleft. Moreover, phosphorylation is known from previous studies to promote E2 competent conformations for Ub charging, inducing electrostatic repulsion and acting on the L7 acidic residues. Here we show that these active conformations are stabilized by a network of hydrophobic interactions between L7 and L4, the latter being a conserved interface for E3-recruitment in several E2s. In the successive steps, L7 conserved acidic residues also provide an interaction interface for both Ub and the Rbx1 RING subdomain of the cognate E3. Our data therefore suggest a crucial role for L7 of family 3 E2s in all the E2-mediated steps of the ubiquitination cascade. Its different functions are exploited thank to its conserved hydrophobic and acidic residues in a finely orchestrate mechanism.     

RING‐between‐RINGs—keeping the safety on loaded guns

EMBO J (2012) 31 19, 3833–3844 doi:10.1038/emboj.2012.217; published online September072012EMBO Rep (2012) 13 9, 840–846 doi:10.1038/embor.2012.105; published online September072012The ‘RING-between-RING''-type E3 ubiquitin ligase HOIP acts via a novel RING/HECT-hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non-covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto-inhibited. This explains why assembly of either HOIP/HOIL-1L or HOIP/SHARPIN is required to catalyse linear chain formation.Post-translational modification of a protein with Ubiquitin (Ub) requires the activity of three enzymes: a Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub ligase (E3). Final Ub transfer is performed by an E3 enzyme, which mediates the ligation of Ub from an E2∼Ub conjugate (‘∼'' denotes a thioester) onto a substrate. E3s are commonly divided into two mechanistic classes: RING/U-box E3s and HECT E3s. RING/U-box E3s facilitate the transfer of Ub from the E2∼Ub directly onto a substrate amino group. In contrast, HECTs transfer Ub from the E2∼Ub to the substrate via a HECT∼Ub intermediate. This mechanistic difference leads to an important distinction regarding what determines the type of Ub product (i.e., the specific Ub-chain linkage) formed: in ubiquitination pathways involving RING-type E3 ligases, the E2 determines the product formed, whereas for HECT-catalysed pathways, the E3 governs product formation (Christensen et al, 2007; Kim and Huibregtse, 2009).RING-between-RING (RBR) E3s comprise a class of E3s that appear to have special properties. Although RBR E3s have been considered as a subfamily of RING E3s, the RBR E3 HHARI (Human Homologue of ARIadne) was recently shown to form a HECT-like E3∼Ub intermediate (Wenzel et al, 2011). Two other members of the RBR family, HOIL-1 and HOIP, form the Linear Ub Chain Assembly Complex (LUBAC), the only E3 ligase known to catalyse the synthesis of linear Ub chains (Kirisako et al, 2006). Linear Ub chains are produced by head-to-tail conjugation of Ub molecules through their N- and C-termini and have been shown to activate the canonical NF-κB pathway (Tokunaga et al, 2009).Two studies by the Rittinger and Sixma groups now reveal important insights regarding the formation of linear Ub chains by the dimeric RBR E3 complex HOIP/HOIL-1L (Smit et al, 2012; Stieglitz et al, 2012). Results from these studies highlight three emerging themes among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions.The RBR E3 ligase domain consists of two distinct RING domains, called RING1 and RING2, connected by an IBR (In-Between-Ring) domain. Despite its name, RING2 is not a canonical RING domain as it contains an active site Cysteine (Cys), which has recently been shown to form a thioester E3∼Ub intermediate, as directly detected for the RBR E3 HHARI. Although the Ub-loaded species could not be detected for the RBR E3 parkin, mutation of the analogous cysteine residue abrogated parkin''s ligase activity implying that it works via the same mechanism. On the basis of these observations, Wenzel et al (2011) proposed that the RBR E3s are a family of RING/HECT hybrids that use RING1 to bind an E2 (RING-like) and RING2 to present the active site Cys (HECT-like) as shown schematically in Figure 1. Both Smit et al (2012) and Stieglitz et al (2012) observed a HOIP∼Ub thioester, confirming that HOIP also acts via a RING/HECT-hybrid mechanism. Furthermore, Smit et al (2012) used a clever strategy to uncouple the first transfer event (E2∼Ub to E3) from the final transfer event (E3∼Ub to substrate Ub) to verify that the E3∼Ub intermediate is a prerequisite for Ub transfer onto a substrate and not just a serendipitous side product. The results extend the number of RBR E3s for which a thioester intermediate has been observed and support the notion that RBR E3s are indeed RING/HECT hybrids.Open in a separate windowFigure 1Three common themes are emerging among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions. RBR E3s are characterized by their RBR domain that consists of two distinct RING domains, RING1 that binds the E2, and RING2 that harbours the active site Cys. Two new studies on the RBR E3 HOIP show that (a) domain(s) in HOIP''s N-terminal region inhibits its ligase activity and (b) a domain C-terminal to HOIP''s RBR binds and orients an acceptor Ub to direct linear Ub-chain formation (‘Linear Ub chain Determining Domain'' or LDD). (A)Three ways in which auto-inhibition might occur are illustrated: (1) inhibition of E2∼Ub binding by RING1, (2) obstruction of the active site cysteine on RING2, and/or (3) occlusion of acceptor Ub binding on the LDD. (B) A possible flow of events that occur once auto-inhibition released is shown. Details of each step and how specifically auto-inhibition is released are still unknown.Previous studies have established that HOIP Ub ligase activity and subsequent activation of NF-κB require either the RBR-containing protein, HOIL-1L, or SHARPIN, an adaptor protein associated with LUBAC (Ikeda et al, 2011; Tokunaga et al, 2011). The two current studies now show that although full-length HOIP exhibits very low activity on its own, removal of the N-terminal ∼700 residues results in robust ligase activity. Thus, HOIP appears to be auto-inhibited in the absence of a binding partner. Further analysis revealed that HOIP''s UBA (Ub-Associated) domain is partly responsible for auto-inhibition, although additional N-terminal domains appear to have auto-inhibitory effects as well. SHARPIN, which contains a UBL (Ub-Like) domain, can relieve auto-inhibition of HOIP. Similarly, the addition of the HOIL-1L UBL domain, previously shown to interact with the HOIP UBA domain (Yagi et al, 2012), relieves inhibition. Interestingly, the addition of full-length HOIL-1L results in even greater ubiquitination activity.Stieglitz et al (2012) show that the RBR E3 HOIL-1L has very low E3 activity on its own. Intriguingly, they found that mutation of the HOIL-1L RING2 active site Cys (C460A) reduced activity of the HOIP/HOIL-1L complex back to levels comparable to HOIP activity in presence of HOIL UBL alone. This suggests a more active, catalytic role for HOIL-1L in linear Ub-chain formation than previously appreciated. The details regarding this role must await further studies, but involvement of an active site Cys residue on a second RING2 domain suggests a possible reciprocal transfer mechanism. Perhaps linear chains can be pre-built via such a mechanism and passed en bloc to substrate, similarly to mechanisms used by some HECT-type bacterial E3 ligases (Levin et al, 2010).Parkin, another RBR E3, also exhibits auto-inhibition (Chaugule et al, 2011), but the auto-inhibitory mechanism and the release thereof differ from HOIP. Unlike parkin''s N-terminal UBL, which is thought to interact within the RBR domain at RING2, HOIP''s UBA does not bind detectably in trans to any region in the RBR domain (Stieglitz et al, 2012). Furthermore, addition of its UBA in trans does not inhibit the activity of HOIP RBR E3 as was seen with parkin and its UBL domain. The auto-inhibition of parkin is likely released by substrate binding, because addition of either the UIM of Eps15 or the SH3 domain of endophilin-A, both known to bind the parkin UBL, can restore the activity of parkin (Chaugule et al, 2011). In addition, phosphorylation of Ser65 within the UBL of parkin by PINK-1 activates parkin, presumably by releasing the UBL from RING2 (Kondapalli et al, 2012). In contrast, HOIP overcomes its auto-inhibition through binding either HOIL-1L or SHARPIN. There is no additive effect when both binding partners are present, consistent with the notion that both proteins act via their UBL domains, although this remains to be demonstrated for SHARPIN. The activity of either SHARPIN/HOIP or HOIL-1L/HOIP can activate NF-κB (Ikeda et al, 2011; Tokunaga et al, 2011), but how the protein complexes differ in their cellular roles remains to be further analysed.The finding that HOIP and parkin exhibit auto-inhibition raises the question whether there is something special about the RBR E3s that require auto-inhibition. In this regard, we note that RBR E3s bind the E2 UbcH7 with significantly tighter affinity than canonical RING E3s bind their E2s (Dove and Klevit, unpublished). In the absence of a substrate, RING1 loaded with UbcH7∼Ub would lead to non-productive transfer of Ub from UbcH7∼Ub to the active site of RING2. Occlusion of the active site by auto-inhibition may therefore act as a safety check until its activity is required for transfer of Ub to a substrate. As yet, there is no evidence to indicate whether substrate binding will release HOIP auto-inhibition, as it does for parkin, but this remains a possibility.The revelation that removal of all domains N-terminal to the HOIP RING1 domain yields a highly active ligase allowed both groups to explore questions pertaining to how linear chains are built. Remarkably, constructs comprised of only the RBR domain through the C-terminus of HOIP are sufficient to specify linear Ub chains. (The two groups use HOIP constructs that differ by only two N-terminal residues (697/699–1072) but Stieglitz et al call their construct RBR whereas Smit et al call it RBR-LDD.) (Smit et al, 2012; Stieglitz et al, 2012). Smit et al (2012) demonstrate that the region immediately C-terminal to RING2 is required for linear chain building activity and name the region the ‘LDD'' (Linear Ub chain Determining Domain). Their results indicate that the LDD binds and orients the acceptor Ub to promote transfer of the donor Ub from the RING2 active site to the N-terminus of the acceptor Ub (Figure 1). Parkin has also been suggested to bind free Ub. Details about whether parkin binds acceptor or donor Ub and whether Ub binding determines Ub-chain specificity are still unknown.There is precedence for acceptor Ub binding by HECT E3s and this interaction is essential for chain formation by NEDD4 and its yeast orthologue Rsp5 (Kim et al, 2011; Maspero et al, 2011). In another example, the inactive E2 variant MMS2 binds an acceptor Ub and orients the Ub-Lys63 into the active site of Ubc13 thereby guaranteeing K63-linked chain formation by the E2 (Eddins et al, 2006). Besides proper orientation of the acceptor Ub, chemical differences between α- and ɛ-amino groups likely contribute to linear Ub-chain specificity. For example, E2s known to be active with RING-type E3s can transfer Ub onto the amino acid lysine, but not the other amino acids containing α-amino groups indicating specificity towards the ɛ-amino of lysine (Wenzel et al, 2011).Catalysed by the unexpected discovery that HHARI is a HECT/RING hybrid E3, details about how the RBR class of E3s function are beginning to emerge. We now know, either directly or indirectly, that at least 4 RBR E3s of the 13 identified in humans (HHARI, HOIL, HOIP, and parkin) require a trans-thiolation event using an active site cysteine within RING2. Conservation of this cysteine among all RBR E3s strongly suggests that the RING/HECT-hybrid mechanism is conserved and therefore defines the class. The hybrid mechanism also offers an explanation for the heretofore puzzling observation that, despite being categorized as a RING E3, HOIP determines the type of Ub chain formed. The ability to bind an acceptor Ub close to the RING2 active site likely contributes to how the RBR E3s dictate the type of product they produce. Finally, both HOIP and parkin are auto-inhibited. It remains to be seen whether HOIP''s auto-inhibitory domains work via inhibition of E2∼Ub binding by RING1, obstruction of the active site cysteine on RING2, and/or occlusion of acceptor Ub binding on the LDD (Figure 1). Regardless of the mechanistic details, the ability to modulate their activity may be a common trait of the RBR E3s. Given recent rapid progress, our understanding of this special class of E3s will continue to grow apace.