Ubiquitination is a central process affecting all facets of cellular signalling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4,5,6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available8,9,10,11,12,13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012)
Metzger, M. B., Pruneda, J. N., Klevit, R. E. & Weissman, A. M. RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim. Biophys. Acta 1843, 47–60 (2014)
Berndsen, C. E. & Wolberger, C. New insights into ubiquitin E3 ligase mechanism. Nature Struct. Mol. Biol . 21, 301–307 (2014)
Wenzel, D. M., Lissounov, A., Brzovic, P. S. & Klevit, R. E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011)
Smit, J. J. et al. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 31, 3833–3844 (2012)
Stieglitz, B., Morris-Davies, A. C., Koliopoulos, M. G., Christodoulou, E. & Rittinger, K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 13, 840–846 (2012)
Kirisako, T. et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006)
Riley, B. E. et al. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nature Commun. 4, 1982 (2013)
Trempe, J. F. et al. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340, 1451–1455 (2013)
Wauer, T. & Komander, D. Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J. 32, 2099–2112 (2013)
Duda, D. M. et al. Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism. Structure 21, 1030–1041 (2013)
Kumar, A. et al. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J. 4, 2506–2521 (2015)
Wauer, T., Simicek, M., Schubert, A. & Komander, D. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370–374 (2015)
Stieglitz, B. et al. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP. Nature 503, 422–426 (2013)
Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011)
Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011)
Tokunaga, F. et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011)
Yagi, H. et al. A non-canonical UBA-UBL interaction forms the linear-ubiquitin-chain assembly complex. EMBO Rep. 13, 462–468 (2012)
Sasaki, K. & Iwai, K. Roles of linear ubiquitinylation, a crucial regulator of NF-κB and cell death, in the immune system. Immunol. Rev. 266, 175–189 (2015)
Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biol. 11, 123–132 (2009)
Plechanovová, A., Jaffray, E. G., Tatham, M. H., Naismith, J. H. & Hay, R. T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012)
Mace, P. D. et al. Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment. J. Biol. Chem. 283, 31633–31640 (2008)
Plechanovová, A. et al. Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nature Struct. Mol. Biol . 18, 1052–1059 (2011)
Dou, H., Buetow, L., Sibbet, G. J., Cameron, K. & Huang, D. T. BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer. Nature Struct. Mol. Biol . 19, 876–883 (2012)
Zheng, N., Wang, P., Jeffrey, P. D. & Pavletich, N. P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102, 533–539 (2000)
Pruneda, J. N. et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012)
Branigan, E., Plechanovova, A., Jaffray, E. G., Naismith, J. H. & Hay, R. T. Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains. Nature Struct. Mol. Biol. 22, 597–602 (2015)
Kamadurai, H. B. et al. Insights into ubiquitin transfer cascades from a structure of a UbcH5B~ubiquitin-HECT(NEDD4L) complex. Mol. Cell 36, 1095–1102 (2009)
Huang, L. et al. Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2–E3 enzyme cascade. Science 286, 1321–1326 (1999)
Luna-Vargas, M. P. et al. Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases. J. Struct. Biol. 175, 113–119 (2011)
Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W. & Klevit, R. E. A. UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol. Cell 21, 873–880 (2006)
Hilgart, M. C. et al. Automated sample-scanning methods for radiation damage mitigation and diffraction-based centering of macromolecular crystals. J. Synchrotron Radiat. 18, 717–722 (2011)
Stepanov, S. et al. JBluIce-EPICS control system for macromolecular crystallography. Acta Crystallogr. D 67, 176–188 (2011)
Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Sakata, E. et al. Crystal structure of UbcH5b~ubiquitin intermediate: insight into the formation of the self-assembled E2~Ub conjugates. Structure 18, 138–147 (2010)
Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000)
Bunkóczi, G. & Read, R. J. Improvement of molecular-replacement models with Sculptor. Acta Crystallogr. D 67, 303–312 (2011)
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
Nicholls, R. A., Long, F. & Murshudov, G. N. Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012)
Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70, 2487–2499 (2014)
Ozkan, E., Yu, H. & Deisenhofer, J. Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. Proc. Natl Acad. Sci. USA 102, 18890–18895 (2005)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006)
Joosten, R. P., Long, F., Murshudov, G. N. & Perrakis, A. The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1, 213–220 (2014)
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr . 66, 12–21 (2010)
Dong, K. C. et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011)
Elliott, P. R. et al. Molecular basis and regulation of OTULIN-LUBAC interaction. Mol. Cell 54, 335–348 (2014)
Spratt, D. E. et al. A molecular explanation for the recessive nature of parkin-linked Parkinson’s disease. Nature Commun. 4, 1983 (2013)
Spratt, D. E., Mercier, P. & Shaw, G. S. Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase. PLoS ONE 8, e74047 (2013)
Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015)
Grishin, A. M. et al. Structural basis for the inhibition of host protein ubiquitination by Shigella effector kinase OspG. Structure 22, 878–888 (2014)
Lehninger, A. L., Nelson, D. L. & Cox, M. M. Principles of Biochemistry . 2nd edn, (Worth Publishers, 1993)
Afonine, P. V. et al. FEM: feature-enhanced map. Acta Crystallogr. D 71, 646–666 (2015)
Emmerich, C. H. et al. Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains. Proc. Natl Acad. Sci. USA 110, 15247–15252 (2013)
Sato, Y. et al. Specific recognition of linear ubiquitin chains by the Npl4 zinc finger (NZF) domain of the HOIL-1L subunit of the linear ubiquitin chain assembly complex. Proc. Natl Acad. Sci. USA 108, 20520–20525 (2011)
The authors thank A. Bobkov (SBP Protein Analysis Facility) for performing ITC and AUC experiments, M. Petroski (SBP) for providing UbcH5B and Cdc34 constructs, J. Badger (DeltaG technologies) for assistance in model evaluation and E. Pasquale (SBP) for help with manuscript writing. This work was supported by NIH grant R01AA017238 and institutional funding (S.J.R.), an EMBO Long-term Postdoctoral Fellowship (B.C.L.), a Rutherford Discovery Fellowship from the New Zealand government administered by the Royal Society of New Zealand (P.D.M.) and NCI Cancer Center Support Grant P30CA030199 (SBP Protein Analysis Core Facility). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006).
The authors declare no competing financial interests.
Extended data figures and tables
a, Domain organization of HOIP as commonly outlined in the literature. HOIP consists of a PNGase/ubiquitin-associated (PUB) domain followed by a B-box zinc-finger (B-box) domain51, NPL4 zinc-fingers (NZF), the auto-inhibitory UBA domain and the RING-between-RING module (RBR, grey background). The HOIP RBR module contains the typical RING1, in-between RING (IBR) and RING2 domains, and a HOIP-specific additional linear ubiquitin chain determining domain (LDD). A yellow circle indicates the RBR catalytic cysteine (C885) forming the HECT-like thioester intermediate with ubiquitin. The binding sites of the other LUBAC constituents HOIL-1L and SHARPIN are also indicated. b, The RBR RING2 domain has the topology of an IBR domain. The individual HOIP RING1, IBR and RING2 domains from the HOIP RBR/E2~Ub/Ub structure are shown to enable direct comparison of their folds. This illustrates that the zinc-finger domain designated RING2 in fact adopts the topology of an IBR, as multiple groups have reported for various RBR E3 ligases previously8,9,10,11,14,52,53. The terms RBR and RING2 however are used in this study for consistency with the widely accepted nomenclature. c, The HOIP RING2–LDD region. HOIP features an extension of its catalytic RING2 domain termed LDD, which adds two zinc-fingers and a helical arrangement to the RING2. The LDD is usually denoted as a domain following RING2. However, Rittinger and colleagues14 showed that the LDD is intertwined with the HOIP RING2 to form a single extended domain that contains a central canonical RBR RING2 with the additional features of the LDD ensuring the linear ubiquitin chain formation characteristic of HOIP. This domain will thus be designated RING2L (for RING2–LDD). The RING2L from the current HOIP structure is displayed with RING2 in light green and LDD in dark green. d, The structural arrangement of active HOIP RBR in the HOIP/E2~ubiquitin complex is markedly different from that of auto-inhibited RBRs. Left, active HOIP RBR from the HOIP/E2~ubiquitin complex. The RING1–IBR region and the RING2L are coloured magenta and green respectively. The individual RBR domains are also highlighted: RING1, yellow circle; IBR, orange circle; RING2, red circle. The RING1 extension helices (hE1, hE2) and IBR–RING2 linker helices (hL1 and hL2) are labelled. Middle and right, analogous representations of auto-inhibited Parkin and HHARI (PDB: 4I1H (ref. 8) and PDB: 4KBL (ref. 11)). Additional domains and regions besides the RBR of Parkin and HHARI are coloured grey.
a, Final 2Fo − Fc (left) and simulated annealing (SA) composite omit (right) electron density maps of select interfaces of the HOIP/UbcH5B~Ub/Ub complex contoured at 1σ. Proteins are shown in sticks and coloured according to Fig. 1. b, Data collection and refinement statistics.
Extended Data Figure 3 Complexity of the crystallographic asymmetric unit and structure of the HOIP/UbcH5B~ubiquitin/ubiquitin E2-E3 transfer complex.
a, The asymmetric unit contains two transfer complexes. Left, colour schematic of the proteins present in the asymmetric unit. Middle, structure of the asymmetric unit. The asymmetric unit contains two HOIP RBR/UbcH5B~ubiquitin complex arrangements (complex 1, 2). The two UbcH5B~ubiquitin conjugates are coloured orange and cyan, respectively, and are bound to two HOIP RBR molecules (magenta and green), which cross over between the complexes. Additional allosteric ubiquitin (Uballo, blue) and UbcH5B~ubiquitin (UbcH5B~Uballo, yellow and blue, respectively) molecules are bound to the HOIP RBRs in complex 1 and complex 2, respectively. Since Uballo makes all contacts with the RBRs and the UbcH5B of the UbcH5B~Uballo conjugate solely mediates crystal contacts (bottom left), only Uballo of complex 1 is displayed and discussed in the text and figures in terms of the additional ubiquitin binding. The black oval indicates an additional HOIP hL1/Ubact inter-complex interaction discussed in panels e and f. Right, close-up of the region where the two RBRs of HOIP cross over between the complexes. The close-up shows that residues D852 and P853 of the respective RBRs come in 6 Å proximity suggesting a continuity in the biological complex in which the two residues from the respective RBRs are linked (as indicated by the grey background), resulting in the monomeric complex schematically illustrated underneath and discussed in panels c–f. b, The RING1–IBR and RING2L form two distinct entities to bind E2~ubiquitin. The monomeric complex as displayed for complex 1 assumes a flexible linkage between the autonomous units of the RING1–IBR arm and the RING2L from the two different RBR molecules in the asymmetric unit. This linkage is formed by residues D852 and P853 connecting IBR and RING2L (schematically illustrated in the cartoon). To test the structural integrity of the assumed link and the autonomy of the RING1–IBR arm on one side and the RING2L on the other side, we introduced a spacer comprising five alanine residues between D852 and P853 in HOIP RBR (see cartoon) and measured the activity of the RBR D852-Ala5-P853 insertion mutant in polyubiquitination assays. The assays show that the mutant (right) retains an activity similar to the wild-type RBR (left) indicating that indeed RING1–IBR and RING2L act as autonomous units. The dramatically reduced activity of HOIP RING2L alone (residues P853 to end) is also shown for reference (middle). c, The HOIP RBR/UbcH5B~ubiquitin complex is monomeric at concentrations of 1.25–5 μM. To determine if the HOIP RBR/UbcH5B~ubiquitin complex is indeed monomeric in solution, we analysed the isolated HOIP RBR/UbcH5B~ubiquitin complex protein material that was used for crystallization by sedimentation equilibrium analytical ultracentrifugation (SE-AUC). SE-AUC provides an absolute, shape-independent measurement of molecular weight, thus allowing accurate determination of the oligomeric state. The three SE-AUC experiments performed on the HOIP RBR/UbcH5B~ubiquitin complex yielded an absolute molecular weight (MW) of 71,658 Da, indicating a monomeric complex. At an order of magnitude higher concentrations (12.5–50 μM), SE-AUC results indicate the formation of a dimer with a MW of ~144 kDa, although curve fitting residuals also show substantial presence of aggregates (data not shown). These results indicate that the biological complex in solution is monomeric at physiological low μM concentrations such as those used for the thioester transfer assays and polyubiquitination assays. However, the dimeric arrangement observed in the crystal structure might be relevant in a high concentration setting such as within the LUBAC complex. Here, a high local concentration of HOIP RBR could favour binding of the E2~ubiquitin between the RING1–IBR and RING2L of two neighbouring molecules. Importantly, all mechanisms depicted in this article hold true for both the monomeric and dimeric states (as illustrated in d). This means that the deduced mechanism is in principle applicable to different RBR E3 ligases of which some might function as dimers in local high concentration assemblies (such as within the LUBAC), whereas others might be active in a monomeric setting. d, Schematic illustration of the dimeric arrangement as observed in the asymmetric unit. The schematic shows that all features deduced (Figs 1, 2, 3, 4 and Extended Data Figs 4,5,6,7,8,9,10) are also valid for the dimeric case (binding of Uballo is omitted for clarity). e, Asymmetric unit dimer-related interactions between HOIP hL1 and Ubact. The dimeric arrangement contains no additional protein–protein interfaces compared to the monomeric assemblies with the exception of hL1 residues W847, M850 and N851, which in the asymmetric unit contact the activated ubiquitin of the other complex (indicated by an oval in a). f, Mutational analysis of HOIP hL1/Ubact interactions. Mutations of HOIP hL1 residues that interact with Ubact have no effect on thioester transfer activity (Coomassie-stained bands in red), indicating that this ‘trans’ complex interaction is not critical for the RBR mechanism, in line with the model of a monomeric arrangement.
Extended Data Figure 4 The HOIP RING1–IBR applies an altered binding mode compared to classic RING E3s necessitating a HECT-like mechanism.
a, The HOIP RBR RING1 uses an E2 interaction pattern similar to classic RINGs, but which results in a shifted binding. Shown are the details of the RING/E2 interaction in the HOIP RING1RBR/UbcH5B~ubiquitin complex (left), the RNF4 RINGclassic/UbcH5A~ubiquitin complex (middle; PDB: 4AP4 (ref. 21)), and the BIRC7 RINGclassic/UbcH5B~ubiquitin complex (right; PDB: 4AUQ (ref. 24)). The HOIP RBR-type RING1 uses a pattern of hydrophobic residues as the core of the interaction with E2 that is similar to that in classic RING E3 ligases. Subtle differences however support a shifted binding mode (see also Fig. 2b). The main features of the RING and E2 as well as HOIP residues mutated in Fig. 2e–g are displayed in bold. Zinc-finger (ZF) 1 and 2 of the RING domains and the SPA-loop of the E2 containing a conserved Ser-Pro-Ala motif are annotated. For the following panels the same structures and colour codes as in a are used. b, The shift in binding and altered surface residues in HOIP RING1 do not support the composite RING/E2 binding site for activated ubiquitin used by classic RING/E2 complexes. UbcH5A/B E2s are rendered as surface representation and the RING domains in ribbon representation. Residues crucial for classic RING E3s to recruit the activated ubiquitin in the composite RING/E2 ubiquitin-binding surface21,24 are depicted (middle, right). In HOIP RING1 (left) equivalent residues are not conserved (displayed), indicating that HOIP cannot accommodate the activated ubiquitin in its RING1/UbcH5B complex. For illustration purposes, only one monomer of the dimeric RNF4 is shown (although Y193 from the other RING molecule is still displayed)21; for BIRC7 the RING dimer (RING2) is displayed24. c, Alignment of HOIP, Parkin and HHARI RING1 domains with classic RING domains centred around residues displayed in b. Residues crucial for ubiquitin binding in classic RINGs are highlighted in cyan and their structural equivalents in the RING1 domains of the RBR E3 ligases HOIP, Parkin and HHARI are indicated by boxes, attesting to the absence of a composite RING1/E2 ubiquitin-binding site in RBR ligases (asterisk indicates ubiquitin interaction residues from the other RING molecule in the dimers formed by the classic RINGs RNF4 and BIRC7). The T/I-C-R sequence observed in the dimeric RINGs of RNF4 and BIRC7 represents the highly conserved Φ-x-R/K motif, where Φ is a hydrophobic residue and x is either a Cys in RING E3 ligases or a polar residue in U-box ligases26. This motif is not only critical for E3-mediated catalysis by dimeric RING ligases (such as RNF4 or BIRC7) but is also necessary for E2-mediated catalysis by simpler monomeric RING and U-Box E3 ligases26. The fact that this motif is not conserved in HOIP, HHARI and Parkin further confirms the mechanistic differences between RBRs and classic RING domains. d. Thioester transfer assays show that E2 residues critical for classic RING-supported catalysis are not important for HOIP catalysis. Left, HOIP RBR thioester transfer assays show similar activity of wild-type UbcH5B and L104A and S108A mutants. This is in stark contrast to the reported effects of these mutations on classic RING-supported catalysis21,24, underlying the fundamentally different mechanism of the HECT-like catalysis by HOIP RBR. Right, mutation to Ala of HOIP RBR N909, which would be in the vicinity of the activated ubiquitin if the E2~ubiquitin conjugate were bound in a bent manner (see e), also shows no effect on HOIP thioester formation (Coomassie-stained bands in red). e, The altered E2~ubiquitin binding mode of RBR RING1 results in the requirement for a HECT-like mechanism. Displayed are the entire RING/UbcH5~ubiquitin complexes with RINGs and E2 s depicted in ribbon representation and the activated ubiquitin in surface representation. The bipartite binding mode used by the HOIP RING1–hE2–IBR arm (see also Fig. 2a) results in an elongated E2~ubiquitin conformation (left, only the RING1 domain of HOIP is depicted) while formation of a composite RING/E2 binding surface in the case of classic RING E3 ligases (middle, right) results in binding of the activated ubiquitin in a compact manner with a bent E2~ubiquitin conformation. Importantly, this bent conformation places the thioester link in a specific position relative to the catalytic machinery of the E2, allowing direct attack by the lysine/amine function of a substrate or growing ubiquitin chain. The Lys85/Ser85 residues mediating the E2~ubiquitin linkage and mimicking UbcH5A/B catalytic cysteine C85 are displayed as red spheres. In the elongated E2~ubiquitin conformation propagated by the HOIP RBR, this attack is not possible. The linkage is however ideally positioned for the attack by the RBR catalytic cysteine in a HECT-like mechanism (see also Fig. 3 and Extended Data Fig. 7a). f, Close-up of the catalytic centres in E2~ubiquitin linkages. Details of the catalytic centres resulting from the E2~ubiquitin conjugate conformations outlined in e and, for comparison, the HECT-type E3 NEDD4L/UbcH5B~Ub structure (PDB: 3JW0 (ref. 28)), with the directionality of an attacking amine indicated as previously proposed21. In the HECT-like RBR arrangement, the UbcH5B~ubiquitin linkage is not aligned correctly relative to the E2 catalytic machinery for a direct attack by an amine function. This is similar in the HECT-type arrangement in the NEDD4L complex but completely different from the arrangement in classic RING-supported E2 catalysis. Additionally, the ubiquitin C-terminal residues G75-G76 reside in a position that would overlap with the attacking amine. The available structure of the BIRC7/UbcH5B~Ub complex (PDB: 4AUQ (ref. 24)) features an UbcH5B N77A mutant and the remainder of the Asn side-chain has been manually added based on wild-type UbcH5B from PDB: 2ESK (ref. 45) (right).
Extended Data Figure 5 Helix–IBR-fold motifs constitute new ubiquitin-binding regions (UBR) in active RBR proteins: binding of the activated ubiquitin by UBR1 using binding mode 1.
a, Schematic illustrating binding mode 1 used by hE2–IBR to bind the activated ubiquitin in the RING1–IBR arm. The general principle of this binding mode is that the RING1 extension helix 2 (hE2) preceding the IBR presents a pattern of charged/polar residues (indicated by blue and red squares, which symbolize K, R, H and E, Q, N residues respectively) that interact with ubiquitin E34 and K11. These interactions are supported by the IBR surface, with a particular contribution of hydrophobic residues (yellow square) flanking the salt bridge system. b, Coordination of the activated ubiquitin by HOIP hE2–IBR in mode 1. HOIP hE2 residues K783 and E787 bind ubiquitin residues E34 and K11 and are flanked by hydrophobic residues M791 and W798 from HOIP IBR. c, Structurally equivalent residues in Parkin and HHARI. Displayed are the hE2–IBR modules from auto-inhibited Parkin (PDB: 5C1Z (ref. 12)) and HHARI (PDB: 4KBL (ref. 11)) with residues equivalent to HOIP residues in b depicted, illustrating the general conservation of UBR1. It should be noted that these structures feature auto-inhibited forms of the RBR proteins, which exhibit a kink in hE2 of UBR1. This kink would sterically hinder ubiquitin binding to UBR1 and probably participates in the RBR auto-inhibition mechanism (see also Extended Data Fig. 8). d, Thioester-transfer assays of ubiquitin and UBR1 salt bridge mutants. In agreement with the observed four-residue salt bridge system in b, the single K11A or E34A ubiquitin mutations show only a slight to moderate effect since the remaining charged residue can still coordinate the two oppositely charged residues of HOIP. In contrast, elimination of both similarly charged residues in the complex by combining the HOIP K783A and ubiquitin K11A or HOIP E787A and ubiquitin E34A mutations results in a more dramatic loss of activity (mean activity ± s.e.m. (n = 3), one-way ANOVA followed by Tukey’s post hoc test; **P < 0.01; ***P < 0.001; NS, not significant; representative gels shown in Supplementary Fig. 1). e, f, Role of the IBR in UBR1. Close-up of the additional IBR/Ubact interactions in stick representation (e) shows that HOIP S803 and ubiquitin K6 coordinate the backbone carbonyl functions of ubiquitin T12 and HOIP A800/K829, respectively. W798, which is involved in hydrophobic interactions, is also displayed in sphere representation. Quantitative thioester transfer assays (f) show that alanine mutants of residues outlined in e cause a significant loss of activity (mean activity ± s.e.m. (n = 3), left: one-way ANOVA followed by Tukey’s post hoc test, right: two-tailed unpaired Student’s t-test; **P < 0.01; ***P < 0.001; representative gels shown in Supplementary Fig. 1).
Extended Data Figure 6 Binding of the activated ubiquitin by UBR2 using binding mode 2 and exclusive binding of E2 and acceptor ubiquitin.
a, Schematic illustrating binding mode 2 used by a helix–IBR-fold motif (hL2–RING2) to bind the activated ubiquitin and position the thioester linkage for the transfer reaction. The second helix (hL2) of the linker between the IBR domain and the catalytic RING2 domain uses a pattern of two or three hydrophobic residues (yellow squares) to interact with the ubiquitin canonical hydrophobic patch surrounding I44 (ref. 28; not shown). Hydrophobic residues of the RING2 IBR-fold complete the hydrophobic interaction network by coordinating residues L71 and L73 in the second hydrophobic patch28 of ubiquitin (not shown). The central hallmark of this binding mode is the coordination of the characteristic di-Arg (R72, R74) motif in the ubiquitin C terminus, resulting in a firm placement of the C terminus on ZF1 of RING2. b, Structure of the interaction of the helix–IBR-fold in HOIP hL2–RING2 (UBR2) with the activated ubiquitin. Left, hL2 residues L860, Y863 and L864 (yellow spheres) interact with ubiquitin residues L8, I44 and V70 (not shown). Additionally, hydrophobic residues F876 and Y878 (yellow spheres) from the IBR-fold of the minimal catalytic RING2 (light green, see also Extended Data Fig. 1c) coordinate ubiquitin residues L8, L71 and L73 (not shown). Right, display of the full HOIP RING2 including the LDD insertion (RING2L). The coordination of the ubiquitin di-Arg motif is achieved by HOIP residues D983 and E976 from the LDD insertion that is part of the catalytic HOIP RING2L (dark green). This results in the placement of the E2~Ub thioester linkage (K85 replacing UbcH5B C85 is shown as orange sticks) in the vicinity of the catalytic HOIP C885. Bottom, alternatively oriented views of the interaction. c, The two hydrophobic patches of ubiquitin engaged by UBR2. The hydrophobic residues of HOIP RING2 interacting with ubiquitin as highlighted in b are shown as yellow sticks. The interaction residues on the canonical hydrophobic patch of ubiquitin (L8, I44, V70) and the second hydrophobic patch (L71, L73)28 are displayed as grey spheres. d, The RING2 domains of Parkin and HHARI also contain a helix–IBR-fold (hL2–RING2) module with patterns of residues consistent with the formation of a UBR2. Left, helical predictions for the region preceding the RING2 domains of HOIP, Parkin and HHARI. The structures of Parkin and HHARI in their auto-inhibited forms do not display a helix equivalent to hL2 because this region is either not defined (in the crystal structures of HHARI and most Parkin structures) or adopts an extended conformation (in two other Parkin structures)8,9,10,11. However, a helical prediction reliability score (with 1 lowest to 9 highest score) calculated using JPred454 shows a strong helical probability for the segment of Parkin and HHARI preceding the RING2 domain. In fact, the score is similar to that of HOIP, which is displayed with the observed helical secondary structure, pointing to the presence of an equivalent of hL2 in active forms of Parkin and HHARI. These RBR E3 ligases also contain residues capable of interacting with the hydrophobic patch in ubiquitin in positions equivalent to HOIP Y863 and L864 (highlighted in yellow). Right, structures of the RING2 domains of PARKIN (PDB: 4I1H (ref. 8)) and HHARI (PDB: 4KBL (ref. 11)) showing hydrophobic residues (yellow sphere representation) in structurally equivalent positions to HOIP F876 and Y878 and residues (labelled red) capable of interacting with the di-Arg motif in their catalytic RING2. Helix hL2 with the conserved hydrophobic residues not present in the crystal structures as discussed above is indicated schematically. Bottom, different orientations with the putative placement of the ubiquitin C terminus indicated schematically. e, The effect of UBR2 alanine mutations in thioester transfer assays increases with their proximity to the di-Arg motif. Left, mutation of HOIP UBR2 hydrophobic residues to alanine. The hL2 L860A and L864A mutations show little effect on activity, while the Y863A mutation and particularly the RING2L F876 and Y878 mutations, which reside proximal to the di-Arg binding motif formed by D983 and E976 (see also Extended Data Fig. 7) show a marked reduction in activity. Middle, right, mutation of complementary ubiquitin residues involved in UBR2 binding. Similarly to the HOIP mutations, the ubiquitin I44A, L71A and L73A mutations show increasing effects with a closer location to the di-Arg motif. Furthermore, the ubiquitin R74A mutation shows a strong effect on activity, emphasizing the importance of its interaction with HOIP and of the resulting placement of the ubiquitin C terminus linked to the E2. The ubiquitin R72A mutant failed to form an UbcH5B~ubiquitin conjugate, thus preventing analysis. Coomassie-stained bands are in red. f, Overlap of the UbcH5B binding site on HOIP RING2L with the binding site of the acceptor ubiquitin. Left, UbcH5B~ubiquitinact (orange/cyan) interaction with RING2L (green) from the HOIP RBR/E2~ubiquitin complex. Right, RING2L interaction with two ubiquitin molecules arranged in linear fashion, mimicking the HOIP RING2L~ubiquitindonor to ubiquitinacceptor (Ubdon, Ubacc) transfer complex (PDB: 4LJP (ref. 14)). Despite the fact that the placement of the donor ubiquitin in the 4LJP structure results from a crystal contact, this ubiquitin exhibits a position identical to that of the activated ubiquitin bound to UBR2 in the HOIP RBR/UbcH5B~Ub complex. It should be noted that the UBR2 interaction with hL2 is missing because the RING2L from the crystal neighbour presenting the donor ubiquitin pushes hL2 into a different conformation. Importantly, in the HOIP RBR/UbcH5B~ubiquitin complex (left) the E2 binds RING2L in a region that overlaps with the binding site for the acceptor ubiquitin (Ubacc, dark blue) in the RING2L~ubiquitindonor to ubiquitinacceptor transfer complex (right). This highlights how the E2~ubiquitin conjugate and the acceptor ubiquitin (which is the substrate of the E3 reaction) cannot bind the RBR at the same time, thus making a HECT-like transfer a requirement in the E3 ligase mechanism of RBR proteins.
a, Close-up view of the catalytic centre of the transfer complex shows conservation of the contact conduits. Top left, close-up view of the catalytic centre in the HOIP/UbcH5B~ubiquitin transfer complex. Contact conduits 1 and 2 are highlighted with grey backgrounds. HOIP catalytic cysteine C885 is depicted in sphere representation. K85 replacing the catalytic cysteine (C85) in UbcH5B and ubiquitin G76 are displayed in stick representation, featuring the UbcH5B~ubiquitin linkage. Top right, model of the conduits in a Parkin/UbcH5B~ubiquitin complex. The structure of Parkin RING2 (from auto-inhibited Parkin, PDB: 4I1H (ref. 8)) was overlaid on that of HOIP RING2 indicating equivalent contact conduits. Bottom left, analogous model for HHARI using RING2 from auto-inhibited HHARI (PDB: 4KBL (ref. 11)). Bottom right, model of the conduits in a HOIP/UbcH7~ubiquitin complex. The model was generated from PDB entry 4Q5E (ref. 55) with UbcH7 (with the free catalytic cysteine C86 displayed) overlaid on UbcH5B of the HOIP/UbcH5B~ubiquitin transfer complex. The structure of the HOIP/UbcH5B~ubiquitin transfer complex and the other models depicted indicate a conservation of the contact conduits. Mechanistically, the conduits allow for the RBR catalytic cysteine and the E2 catalytic cysteine~ubiquitin linkage to be in close proximity, which serves as main driving force of the transesterification reaction. A reaction driven mainly by proximity is also in agreement with the chemical nature of the catalytic cysteine, which has a pKa of ~8 for the free amino acid. This allows the cysteine to naturally deprotonate, without an absolute need for HOIP H887 (refs 10, 14), before attack of the ubiquitin G76 carbonyl function. In addition, the thioester linkage is far more labile than for example an amide bond, thus further facilitating a proximity-mediated reaction56. However, in light of the geometric arrangement observed, additional subtle catalytic contributions of H887 in supporting the transition state of the reaction and/or re-protonation of the E2 catalytic cysteine are in principle possible. This prospect is particularly intriguing because UbcH7 exhibits a potential break in conduit 2 (between H887 and H119), yet provides its ‘own’ histidine (H119) to the catalytic centre. b, Thioester transfer assays for HOIP contact conduit mutants. Left, thioester transfer assays show that the D983A and E976A mutations strongly affect activity. This is consistent with D983 forming the di-Arg binding motif in UBR2 (see Extended Data Fig. 6) and E976 bridging the three proteins in the complex. In contrast, the H887A mutation does not have a marked effect, in agreement with published results10,14. The Q974A mutation also does not have a strong effect, pointing to a weak auxiliary function of this residue in support of the critical D983 (Coomassie-stained bands in red). Right, the UbcH5B R90A mutation shows a moderate yet significant effect, in line with the structure, which suggests a more pronounced effect for HOIP E976A than for UbcH5B R90A. Surprisingly, mutation of the catalytic D117 in UbcH5B, which is essential for classic RING-supported catalysis, shows a positive effect on the HECT-like thioester transfer, further emphasizing a separate mechanism for RBR HECT-like catalysis (as outlined in Fig. 2 and Extended Data Fig. 4). The gain of function of the D117A mutation also points to a trade-off for this E2 residue to participate in the classic RING-supported versus RBR HECT-like E2/E3 mechanisms (mean activity ± s.e.m. (n = 3), two-tailed unpaired Student’s t-test; **P < 0.01; representative gels shown in Supplementary Fig. 1) c, Polyubiquitination assays for HOIP contact conduit alanine mutants. These assays show similar activity profiles as the thioester transfer assays except for the H887A mutation, which is essential for amide bond formation in the second transfer reaction10,14.
Extended Data Figure 8 The hE2–IBR module contains an additional UBR (UBR3) that binds an allosteric ubiquitin using binding mode 2.
a, UBR3 binds the allosteric ubiquitin (Uballo) using binding mode 2. Cartoon depicting the overall features of binding mode 2 in UBR3. The binding of Uballo is largely analogous to the binding of the activated ubiquitin by UBR2 in the helix–IBR-fold of hL2–RING2L (Extended Data Fig. 6a). Yellow squares indicate hydrophobic patches. b, Location of the UBR3/Uballo interface in the overall complex and its relation to the UBR1/Ubact interface. The additional ubiquitin (Uballo) binds to UBR3 in the hE2–IBR module immediately across UBR1 and the activated ubiquitin. The electron density map (feature-enhanced map57, grey, contour level σ = 1) for the additional ubiquitin is also depicted on the right. c, Details of the UBR3/Uballo interaction and its similarity to the interaction of phospho-ubiquitin (pUb) with Pediculus humanus Parkin (Ph-Parkin). Top left, close-up of HOIP UBR3. Uballo binds to the hE2–IBR module with additional contacts to hE1. Depicted are hydrophobic residues of HOIP (V789, L790, F796, W798 and I807) interacting with the ubiquitin canonical hydrophobic patch (L8, I44 and V70/not shown) and a second hydrophobic patch (L71 and L73/not shown), with the critical HOIP I807 emphasized. The di-Arg binding motif is also depicted, with E809 coordinating ubiquitin R72 and R74 and aligning the ubiquitin C terminus in a parallel manner with sheet β2 of the IBR. A red arrow indicates that UBR3 sterically allows the binding of di-ubiquitin/polyubiquitin chains on the C-terminal side of the bound ubiquitin (see also Extended Data Fig. 9). Ubiquitin Ser65 is indicated for comparison with the pUb/Ph-Parkin structure (bottom). Top middle, close-up on the di-Arg binding motif. Top right, close-up on the additional contacts between hE1 and Uballo. HOIP R770 interacts with D766, which makes contacts to Y778, and the backbone carbonyl functions of ubiquitin K63 and E64. Bottom, the recent structure of phospho-ubiquitin bound to Ph-Parkin (PDB: 5CAW (ref. 13)) reveals a similar mechanism. Left, close-up with the residues corresponding to those in HOIP depicted. Middle, close-up of the di-Arg motif. Ph-Parkin D346 coordinates R72 similar to HOIP UBR3/Uballo. The chemical tether introduced in the pUb/Ph-Parkin structure between the phospho-ubiquitin C terminus and a non-conserved Cys in Ph-Parkin (C349) shifts ubiquitin R74 away from Ph-Parkin D346 indicating that the di-Arg binding motifs of HOIP and Parkin undergo a similar interaction with ubiquitin or phospho-ubiquitin respectively. Right, the Ph-Parkin interaction equivalent to the HOIP hE1 interaction involves a ubiquitin phospho-serine 65 (pSer65) binding pocket in Ph-Parkin. Phospho-serine 65 is directly coordinated by R307 and Y314 and also H304, which is positioned similarly to HOIP D766. d, The binding of phospho-ubiquitin propagates the formation of UBR1 in Parkin. Overlay of Uballo (slate) bound to HOIP (magenta) and phospho-ubiquitin (light blue) bound to Ph-Parkin (grey blue). Ubiquitin binding propagates a straight conformation of hE2 and an opening of the IBR relative to the extended RING1 (only hE1 is shown). The tether introduced in the phospho-ubiquitin/Ph-Parkin interaction appears to exert a strain on IBR loop 2 and thus the IBR. This suggests that in the absence of the artificial tether phospho-ubiquitin can propagate the formation of a fully functional UBR1 (binding Ubact) in Parkin concomitantly to relieving the UBL autoinhibition, as elegantly demonstrated by Komander and colleagues13. e, The interaction of phospho-ubiquitin with a site analogous to UBR3 in Ph-Parkin causes a straight conformation of hE2 and reorientation of the IBR relative to RING1 as prerequisite to accommodate the activated ubiquitin. Left, full-length auto-inhibited human Parkin (PBD: 5C1Z (ref. 12)). Right, tethered phospho-ubiquitin in complex with ΔUBL-Ph-Parkin (PDB: 5CAW (ref. 13)). f, Conservation of UBR3 in HHARI. HHARI features a helix–IBR module similar to that of HOIP, with conserved hydrophobic patches and a polar residue (Q288) in the position of HOIP E809 that is in principle capable of binding the ubiquitin di-Arg motif. The hE2–IBR region from auto-inhibited HHARI (PDB: 4KBL (ref. 11)) is displayed. As for auto-inhibited Parkin, a key difference from active HOIP is the kink in helix hE2 of auto-inhibited HHARI (see also panel g and Extended Data Fig. 5c). Taken together, these features indicate an overall similarity and the existence of a UBR3 that is allosterically linked to UBR1 in HOIP, Parkin and HHARI and potentially also other RBR proteins (see alignment in Supplementary Data 2). g, HHARI UBA domain (pink) binds HHARI RBR (light orange) in an equivalent position as Uballo binding to HOIP, but promotes an inhibitory conformation of RING1–IBR that cannot bind the activated ubiquitin (PDB: 4KBL (ref. 11)). h, UBR3 is a regulatory hotspot and UBR3/UBR1 crosstalk. Parkin auto-inhibition is facilitated by an UBL domain and is inherently linked to the equivalent of UBR3 and counteraction by phospho-ubiquitin binding. Different to Parkin, no structure of auto-inhibited HOIP RBR is available and thus the conformation of auto-inhibited HOIP is not known. However HHARI is, like HOIP, auto-inhibited by its UBA domain and the auto-inhibited structure of HHARI has been solved previously (PDB: 4KBL (ref. 11)). This structure reveals that unlike the UBL of Parkin, the UBA of HHARI directly utilizes the region of UBR3 for binding and auto-inhibition, which includes the kink in hE2 and a relative RING1–IBR positioning that is incompatible with binding of Ubact as observed in this study, further underlying a regulatory ‘hotspot’ function of the RBR UBR3/UBR1. Left, the UBA domain (pink) of HHARI utilizes an anti-parallel β-sheet anchor with strand β2 of the HHARI IBR (light orange) positioning the UBA to induce a kink in helix hE2 compared to its conformation in active HOIP (magenta) and counteracting the formation of a productive UBR3 and UBR1. Right, binding of the Uballo to UBR3 in mode 2 utilizes a parallel β-sheet anchor (centred around the di-Arg binding interaction) with strand β2 of the IBR in active HOIP (magenta) inducing a straight conformation of hE2 and a conformation of UBR1 suited to bind the activated ubiquitin of the E2~Ubact conjugate. Putative shifts for an analogous UBR1 formed in HHARI are indicated. Of note, the structure of auto-inhibited HOIP is not known and therefore the placement of the UBA in the schematic illustration of Extended Data Fig. 10 is deduced based on the similar auto-inhibition of HOIP and HHARI by their UBA domains, which still needs to be demonstrated.
Extended Data Figure 9 UBR3 interacts with di-ubiquitin and allosterically promotes E2~ubiquitin binding and HOIP RBR activation.
a, ITC experiments analysing the binding of mono-ubiquitin or linear di-ubiquitin to an isolated HOIP RBR/E2~ubiquitin complex. While the binding of mono-ubiquitin is below the sensitivity of the experimental setting (Kd > 50 μM), the binding of linear di-ubiquitin exhibits a Kd of 7.1 μM. b, ITC experiments analysing the binding of UbcH5B~ubiquitin to HOIP RBR in the absence and presence of different di-ubiquitin chains. Top left, the binding stoichiometry of UbcH5B~ubiquitin and wild-type HOIP RBR is n = 1.8, indicating that two UbcH5B~ubiquitin molecules interact with one RBR through UBR1/2 (catalytic binding) and UBR3 (binding of the ubiquitin moiety of the E2~ubiquitin conjugate) with a combined overall Kd of 1.6 μM. The graph shows a single titration step, indicating ‘crosstalk’ between UBR3 and UBR1. Top right, the presence of K48-linked di-ubiquitin leads to 1:1 binding (n = 0.9) of UbcH5B~ubiquitin to HOIP RBR, indicating that di-ubiquitin occupies UBR3 and limits UbcH5B~ubiquitin binding to only its bona fide catalytic binding site (with ubiquitin binding to UBR1/2 and UbcH5B binding to RING1/RING2L). However, the presence of K48-linked di-ubiquitin results in the lowest affinity (Kd = 3.4 μM) for the binding of the conjugate to the RBR, indicating a negative effect of this linkage compared to the other di-ubiquitin entities tested. Bottom left, K63-linked di-ubiquitin has a more favourable effect on UbcH5B~ubiquitin binding (Kd = 2.0 μM), Bottom right, the strongest allosteric effect is observed in the presence of linear di-ubiquitin, which enables sub-micromolar binding (Kd = 600 nM) of UbcH5B~ubiquitin. These results show that linear di-ubiquitin functions as a potent activator of HOIP RBR by binding to UBR3 (see also below and Fig. 4). While the structure depicts the interactions of one ubiquitin unit with UBR3, a second ubiquitin C-terminal to the UBR3-interacting ubiquitin may undergo further interactions with the IBR (as indicated by the arrow in Extended Data Fig. 8c). The cartoon representations summarize the configuration of each ITC experiment. c, Polyubiquitination assays of UBR3 mutants. The HOIP RBR I807A, E809A and R770A mutants exhibit a marked reduction in activity, supporting the importance of UBR3 in HOIP function. d, Activation of HOIP RBR by di-ubiquitin. While wild-type HOIP RBR is activated by the presence of increasing concentrations of wild-type linear di-ubiquitin, wild-type linear di-ubiquitin only has a weak effect on activation of the UBR3 R770A mutant (similar to the I807A and E809A mutants in Fig. 4b). Additionally the di-ubiquitin mutant I44A, which is mutated at a critical UBR3-interacting residue in both ubiquitin units, does not have an activating effect on wild-type HOIP RBR thioester activity (mean activity ± s.e.m. (n = 3), one-way ANOVA followed by Tukey’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; representative gels shown in Supplementary Fig. 1). e, Effect of linear versus K48-linked di-ubiquitin on HOIP RBR thioester transfer activity. In contrast to the ITC binding studies in b, linear and K48-linked di-ubiquitin are both able to increase the thioester transfer activity of HOIP RBR (although the experimental setup necessary to investigate the K48-linkage resulted in larger error; see Methods; mean activity ± s.e.m. (n = 3), one-way ANOVA followed by Tukey’s post hoc test; *P < 0.05; **P < 0.01; NS, not significant; representative gels shown in Supplementary Fig. 1). These results show an UBR3-dependent activating effect of di-ubiquitin, and thus potentially of polyubiquitin chains. However, whether HOIP UBR3 acts as a universal ubiquitin sensor or has a preference for linear ubiquitin over other types of linkage needs to be further examined through careful investigations also including full-length proteins of the LUBAC in cellular settings. Additionally, although there is a substantial gap between UBR3 and the position of the acceptor ubiquitin, longer acceptor ubiquitin chains might be able to bridge this gap and mediate a cooperative effect between the two sites. This would be consistent with a recent publication showing that the presence of K63-linked ubiquitin chains is frequently necessary for the formation of linear polyubiquitin chains58. f, Protein expression levels for the NF-κB reporter assays in cells shown in Fig. 4d. Shown are anti-Flag immunoblots of wild-type HOIP and mutants and anti-myc immunoblots of HOIL-1L, demonstrating similar protein expression levels in different cell lysates. Lysates were also probed by immunoblotting for actin as a loading control. Uncropped blots are shown in Supplementary Fig. 1. g, Time course of HOIP~ubiquitin thioester transfer assay. Left, SDS–PAGE showing time course of HOIP~ubiquitin thioester transfer assay. Coomassie-stained bands in red visualized using LI-COR Odyssey at 700 nm. Right, plot of quantified HOIP~ubiquitin thioester transfer assay time-course (mean ± s.e.m., n = 2). The 10-s time point used in the end-point assays throughout the study is highlighted in red.
HOIP RBR is initially auto-inhibited by its UBA domain. Sequestration of the auto-inhibitory HOIP UBA domain by HOIL-1L5,6,7,18 releases the conformational restraint exerted by the UBA, allowing formation of UBR1 and UBR3. Binding of a ubiquitin entity such as a linear ubiquitin chain to UBR3 stabilizes the active conformation of UBR1 and the RING1–IBR arm, facilitating binding of the E2~ubiquitin conjugate. In the subsequent HOIP/E2~ubiquitin transfer complex, the E2~ubiquitin conjugate is engaged in a clamp-like manner bringing the RBR active cysteine and the E2~ubiquitin thioester in close proximity, ultimately leading to the transfer of the ubiquitin to the RBR cysteine. The E2 then vacates the complex, freeing the site for binding of the acceptor ubiquitin, the N-terminal amine of which attacks the RBR thioester7,14. Once the ubiquitin chain linkage is formed, the ubiquitinated substrate/growing ubiquitin chain must exit RING2L to enable binding of a new E2~ubiquitin conjugate for the next loading of the RBR in the HECT-like E3 ligase cycle. The growing ubiquitin chain could be retained near the RBR by the HOIP NZF domains, HOIL-1L or SHARPIN15,16,17,59, directly linking the HECT-like mechanism to co-operative processes within the LUBAC.
About this article
Cite this article
Lechtenberg, B., Rajput, A., Sanishvili, R. et al. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation. Nature 529, 546–550 (2016). https://doi.org/10.1038/nature16511
Single-Domain Antibodies as Crystallization Chaperones to Enable Structure-Based Inhibitor Development for RBR E3 Ubiquitin Ligases
Cell Chemical Biology (2020)
Nature Chemical Biology (2020)
Journal of Biomolecular NMR (2020)
Genome Instability & Disease (2020)
Pharmacological Reviews (2020)