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focus on ubiquitin
New insights into ubiquitin E3 ligase mechanism Christopher E Berndsen1 & Cynthia Wolberger2,3
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E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated. The attachment of the small protein ubiquitin to substrates regulates a vast array of processes in eukaryotes1,2. Substrates can be modified with a single ubiquitin (Ub) or with a polyubiquitin chain in which one ubiquitin is conjugated to the next3. Ubiquitin is covalently attached to substrate lysines in a three-enzyme cascade catalyzed by E1, E2 and E3 enzymes4, thus resulting in an isopeptide linkage between the ubiquitin C terminus and the ε-amino group of lysine (Box 1). Ubiquitin itself can be ubiquitinated at any one of its seven lysines to give rise to different types of polyubiquitin chains3. Recent studies have shown that ubiquitin can also be covalently linked to N-terminal α-amino groups via a peptide linkage to yield linear polyubiquitin chains5 or N-terminal ubiquitin fusions6,7. Ubiquitin is activated in an ATP-dependent reaction by the E1 enzyme, which forms a thioester with the ubiquitin C terminus, and is then transferred to the active site cysteine of the E2 enzyme to yield an E2~Ub thioester inter mediate (Box 1). The E3 ligase binds to both the E2~Ub thioester and the substrate, catalyzing transfer of the ubiquitin from the active site cysteine of the E2 to the substrate lysine or N terminus8. E3 ligases thus have dual roles as both molecular matchmaker and catalyst, bringing together the right E2 with the right substrate and greatly increasing the rate of ubiquitin transfer. The need to specifically target a broad array of substrates accounts for the great diversity among the estimated >600 human E3s9. Nonetheless, E3 ligases fall into three classes10, each characterized by conserved structural domains and the mechanism by which ubiquitin is transferred from the E2 to the substrate. The really interesting new gene (RING) family catalyzes direct transfer of ubiquitin from the E2 enzyme to the substrate, simultaneously binding both the E2~Ub thioester and the substrate8,11. In contrast, the homology to E6AP C terminus (HECT) and the RING-between-RING (RBR) family E3s ubiquitinate substrates in a two-step reaction in which ubiquitin is transferred from the E2 to an active site cysteine in the E3 and then from the E3 to the substrate12,13. Despite numerous structural 1Department
of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, USA. 2Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 3Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Correspondence should be addressed to C.W. ([email protected]). Received 21 November 2013; accepted 28 January 2014; published online 4 April 2014; doi:10.1038/nsmb.2780
and biochemical studies of ubiquitin conjugation by both E2 and E3 enzymes, the catalytic mechanisms by which E3 ligases enhance rates of ubiquitin transfer have remained remarkably elusive. The mystery of the E3 ligase catalytic mechanism has finally yielded to a series of recent structural and biochemical studies that have given insights into the mechanism by which all three classes of E3 ligases stimulate substrate ubiquitination as well as the way in which E3 ligase activity is regulated. E2 enzymes, essential partners of E3s All E3 ligases bind an E2~Ub thioester and either catalyze transfer of ubiquitin from the E2 to a substrate lysine via an aminolysis reaction, as is the case for RING E3s, or to another cysteine in the E3 via a transthioesterification reaction, as is the case for HECT and RBR E3s. The RING E3 ligases contain no active site residues per se, but they instead greatly enhance the rate of ubiquitin transfer from the E2 active site to the substrate lysine (or N terminus). In this reaction, the amino group in the substrate attacks the thioester linkage between the ubiquitin C terminus and the active site cysteine of the E2, thus leading to hydrolysis of the thioester and formation of an isopeptide (or peptide) bond with the substrate. E2 enzymes contain a highly conserved catalytic domain of ~150 amino acids 14,15. E2 enzymes contain, in addition to the active site cysteine, a conserved asparagine that had been proposed to stabilize an oxyanion reaction intermediate16 but appears to have an essential role in stabilizing the E2 structure in the vicinity of the active site 17. Although there are no other highly conserved residues in the region of the E2 active site, there are typically one or more acidic residues in the vicinity of the active site cysteine that have been proposed to facilitate ubiquitin transfer by orienting and deprotonating the attacking lysine18,19. In the E2 Cdc34, which lacks the corresponding acidic residues, a substrate glutamic acid neighboring the acceptor lysine has been proposed to have an analogous role20,21. In the absence of an E3, the ubiquitin in the E2~Ub thioester is highly mobile, with NMR and crystallographic studies showing multiple conformations of ubiquitin relative to the E2 (refs. 22–25). An important question for understanding all types of E3 ligases was therefore how and whether the donor ubiquitin was positioned for the next step in ubiquitin transfer and how the substrate lysine (in the case of RING E3s) or cysteine (in the case of HECT and RBR E3s) was oriented to attack the E2~Ub thioester linkage.
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Box 1 The ubiquitin cascade and chain formation
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(a) The ubiquitination cascade begins with ATP-dependent charging of the E1 enzyme and results in formation of a thioester bond between the ubiquitin C terminus and the E1 active site cysteine. Ubiquitin is transferred to the E2 active site cysteine in a transthioesterification reaction. An E3 ligase catalyzes transfer of the ubiquitin from the active site cysteine of the E2 to a primary amine on a lysine side chain or protein N terminus. There are three classes of E3 ligases: RING E3s, which bind to both E2~Ub thioester and substrate and catalyze attack of the substrate lysine on the thioester, and HECT and RBR E3s, which both have active site cysteines and catalyze substrate ubiquitination in a two-step reaction involving formation of a thioester with the HECT or RBR E3 followed by attack of the substrate lysine or N terminus on the E3~Ub thioester to form an isopeptide (or peptide) linkage between the ubiquitin C terminus and lysine (or the protein N terminus). (b) Polyubiquitin chains. Ubiquitin (PDB 1UBQ80) has seven lysines and an amino terminus (Met1) that can be ubiquitinated to give rise to different types of polyubiquitin chains. Diubiquitin structures are shown for linear (Met1-linked) (PDB 2W9N81), K63-linked (from PDB 3HM3 (ref. 82)) and K48-linked diubiquitin (1AAR83).
a
ATP
AMP
Ub
SH
S
E1
SH
E1
E1 Ub
Ub SH
S
E2
E2 HECT RBR
RING
Ub Lys
S
E2
Substrate
SH
Ub
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Lys
S E2
Substrate
Ub
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Ub
Ub
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SH E2
Substrate
Ub Lys
SH E2
SH
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Substrate
E3
b K63 Met1
K33
K6
K48
K29
K27
K63
Met1
K48
K11
C
Ubiquitin
302
Linear diubiquitin
K63-linked diubiquitin
K48-linked diubiquitin
Catalysis of ubiquitin transfer by RING E3s RING E3 ligases contain a RING (or RING-like) domain that is responsible for both binding to the E2 and stimulating ubiquitin transfer26. The RING domain adopts a conserved cross-brace structure with two structural Zn2+ ions (Fig. 1a) and can be monomeric27–30 or dimeric25,26,31,32, with dimerization mediated either by the RING domain alone or facilitated by flanking helical regions32–34 (Fig. 1b). A related domain known as the U box has a similar function and fold but has a hydrophobic core in place of the structural metal ions35. RING domains bind to the N-terminal helix of E2 conjugating enzymes30 (Fig. 1c). In the presence or absence of an E2-bound ubiquitin, the E2-E3 interaction is transient and appears to compete with binding of the E1 enzyme to E2 (ref. 36). In addition to the E2-binding domain, RING E3 ligases contain multiple additional domains including those that recruit the substrate for ubiquitin conjugation (reviewed in ref. 8). A subset of RING E3 ligases known as cullin E3s (Fig. 1d) are multisubunit proteins containing a scaffold protein, called the cullin protein, a RING-box protein, containing a domain similar to the RING domain found in single-polypeptide E3 ligases, and a bridging protein, called an F-box protein, for binding the substrate (reviewed in refs. 37,38). Whereas the determinants of RING domain–E2 interactions have been elucidated through studies of many RING domain–E2 complexes27,29,30,34,39,40, these structures provided little insight into the mechanism by which RING domains stimulate ubiquitin transfer. The distance between the RING domain and the E2 active site (Fig. 1c) ruled out direct participation of RING residues in catalysis, and proposals that RING binding communicated allosteric changes to the active site lacked support41. Studies showed that RING domains bind more tightly to E2~Ub conjugates17,42–44, pointing to a role for RING domains in contacting the donor ubiquitin. However, a confounding obstacle was the inability to trap a complex between a RING domain and an E2~Ub thioester for structural studies, because addition of the RING causes rapid hydrolysis, even when a more stable oxyester mimic is used19. This barrier was finally overcome by a clever replacement of the native thioester with an isopeptide linkage, which was done by substituting the active site cysteine of the E2 UbcH5A with a lysine and charging the mutant enzyme with ubi quitin19. In the structure of this E2~Ub conjugate bound to the RING domain of RNF4, Plechanovová et al.19 showed that the RING domain contacts a hydrophobic patch on the donor ubiquitin; this interaction immobilizes the donor ubiquitin and positions its C terminus in a structurally conserved groove in the E2 (Fig. 2a,b). The position of the ubiquitin tail in the RNF4–UbcH5 structure resembles a product complex between the ubiquitin-like protein SUMO and its E2 (ref. 45). In an independent approach used to determine the structure of the BIRC7 RING domain bound to UbcH5B~Ub28, two mutations were used to stabilize the E2~Ub linkage: the active site cysteine was mutated to serine, which forms a more stable ester linkage with ubiquitin, and the conserved asparagine was mutated to alanine (N77A). As with RNF4, both subunits of the dimeric BIRC7 RING domain position the donor ubiquitin attached to UbcH5B. Both of these studies agree with solution NMR experiments showing that the RING heterodimer, BRCA1–BARD1, and the monomeric U box, E4B, restrict the position of the donor ubiquitin on the E2 (ref. 46). Whether this mechanism of conformational selection (Fig. 2c) applies to RING proteins found in the cullin family of ligases has not been demonstrated but is likely, given the similarities in the E2 binding modes. How does conformational selection increase the rate of ubiquitin transfer? Positioning ubiquitin presumably places the reactive thioester between E2 and the ubiquitin C terminus in the correct
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orientation to react with the attacking lysine (Fig. 2d). Immobilizing a bond can increase the rate of reaction47,48 and consequently also account for the increased rate of thioester hydrolysis in the presence of a RING domain49. Additional contributions may come from the correct positioning of the thioester and acceptor lysine in relation to conserved residues that flank the immobilized ubiquitin tail and the active site cysteine (Fig. 2b). These include residues critical for activity, such as acidic residues (Asp117 in UbcH5B) that have been proposed to activate the attacking lysine17,19,28,45, as well as a conserved asparagine (Asn77 in UbcH5B) that has been proposed to stabilize an oxyanion intermediate16 but which was recently shown to have an important structural role in configuring residues in the vicinity of the active site17 (Fig. 2d). Further studies will be needed to elucidate the mechanism by which these residues contribute to catalysis once the donor ubiquitin has been correctly positioned on the E2. Although the RING domain has the primary role in catalyzing ubiquitin transfer, recent studies have shown that some RING E3 ligases contain additional domains that bind to the E2 and influence substrate ubiquitination. The RING E3 gp78 contains an additional helical domain, known as the G2BR50, that binds to the E2 β-sheet on the face opposite that of the active site (Fig. 3a), known as the ‘back side’, to which free ubiquitin has been observed to bind in solution51. G2BR binding is proposed to trigger allosteric changes in the E2 Ube2g2, opening the active site for substrate binding and stimulating ubiquitination50. More recent work shows that RING-domain binding to Ube2g2 antagonizes G2BR binding, promoting E2 enzyme exchange during processive ubiquitination by gp78 (ref. 52). Rad18, the E3 ligase that catalyzes monoubiquitination of PCNA, contains an N-terminal RING domain that binds to the E2 Rad6, as well as a distinct region at the C terminus of the protein53 that contacts
a
b Gln40 Arg181 Ubiquitin
Arg72 Gln92 Asp87
Arg74
RNF4 (RING E3) Leu86 Gly75 UbcH5A (E2)
Asn114 Asn77
Gly76 C85K
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Asp117
d Asn114
Asp117 Ub
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Figure 1 RING E3 ligases. Bound zinc atoms in all structures are depicted as red spheres. (a) Monomeric RING from c-CBL (PDB 1FBV30). (b) RING homodimer from BIRC7 (PDB 4AUQ28). Two α-helices contribute additional dimer contacts. (c) TRAF6 ring bound to the E2 Ubc13 (PDB 3HCT29). The active site cysteine of Ubc13 is depicted as spheres. (d) Cullin RING E3 ligases are multisubunit proteins with separate substrate-binding domains (the F box protein (Fbp)) and RING, which binds the E2. Composite model assembled from PDB 1P22 (ref. 84), 1LDJ85, 3DQV86 and 4AP4 (ref. 19). CTD, C-terminal domain; NTD, N-terminal domain.
Ub E2
Lysine
Gly75 Gly76
Ub Asn77 Cys85
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b
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c
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the E2 back side (Fig. 3b) and suppresses the intrinsic polyubiquitination activity of Rad6 (ref. 39). The Cue1 subunit of yeast ERAD E3 ligase complexes contains a U7BR domain that also binds to the back side of Ubc7 (Fig. 3c) and stimulates both E1 charging and substrate ubiquitination54. Whereas the additional domains in all three of these examples all contact the same surface of the E2, neither the structures nor the mode of interaction is conserved, thus making it difficult to derive any general principles for how these proteins stimulate E2 activity. Understanding the underlying mechanism by which these additional interactions with the E2 contribute to substrate ubiquitination will require determining the somewhat mysterious role of noncovalent ubiquitin binding to the E2 back side51. Two-step ubiquitination by HECT E3 ligases In contrast to RING E3 ligases, which catalyze direct attack of the substrate lysine on the E2~Ub thioester, HECT-domain E3 ligases catalyze two distinct reactions (Box 1): a transthioesterification reaction, in which ubiquitin is transferred from the E2 active site cysteine to a cysteine in the HECT domain, and a subsequent attack on the HECT~Ub thioester by a substrate lysine12. The conserved HECT domain itself comprises an N lobe that binds to the E2 and a Figure 2 Mechanism by which RING E3s stimulate ubiquitin transfer. (a) Structure of RNF4 RING dimer (green, light green) bound to the E2 UbcH5A (blue) charged with ubiquitin (yellow) (PDB 4AP4 (ref. 19)). Omitted from this view is a second E2~Ub conjugate that binds to the light-green RING monomer. (b) Close-up view showing hydrogen-bond interactions between the C-terminal tail of ubiquitin (yellow), E2 (blue) and an arginine in the RNF4 RING (green). The E2 active site cysteine is replaced with a lysine (C85K), which forms an isopeptide bond with the ubiquitin C terminus in the crystallized complex. (c) Activation of E2~Ub for ubiquitin transfer by conformational selection. The thioester-linked ubiquitin is free to adopt multiple positions, but binding of the RING to E2 immobilizes the donor ubiquitin. (d) Model for attack of acceptor lysine (magenta) on the thioester linkage. Asp117 has been proposed to deprotonate the lysine and orient it for nucleophilic attack. Asn77 is in van der Waals contact with the modeled thioester and also forms two conserved hydrogen bonds with backbone atoms.
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Figure 3 E2 back-side binding by additional domains in RING E3 ligases. (a) The E2 Ube2g2 (blue) bound to the G2BR (magenta) and RING (green) of the E3 ligase gp78 (PDB 4LAD52). (b) The Rad6-binding domain (R6BD, magenta) of Rad18 bound to the E2 Rad6 (blue) (PDB 2YBF39). (c) The U7BR (magenta) domain of Cue1 bound to the E2 Ubc7 (blue) (PDB 4JQU54).
b
C lobe that contains the active site cysteine (Fig. 4a). A flexible tether between the N and C lobes permits multiple relative configurations of these two lobes that have proved critical to HECT E3 activity (Fig. 4a). Early structural studies of the E6AP55 and WWP1 (ref. 56) HECT-domain proteins showed the N lobe and C lobe to be distant from each another and suggested that a conformational rearrangement of the N and C lobes was needed to bring the E2~Ub thioester sufficiently close to the active site cysteine in the HECT C lobe. A structure of the HECT E3 NEDD4L bound to a UbcH5B~Ub conjugate57 showed that the C lobe rotated markedly, thus causing the HECT domain to adopt a more compact conformation that brought the E2~Ub thioester near the cysteine in the C lobe and permitted direct contacts between the N lobe and the ubiquitin (Fig. 4b). Although the active site cysteine of NEDD4L is still 8 Å from the cysteine of UbcH5B in this structure57, a modest additional rotation of the C lobe closes the gap sufficiently to permit the transthioesterification reaction to occur. The remaining question of how a charged HECT E3~Ub thioester transfers ubiquitin to a substrate lysine was addressed only recently by two structural studies of Nedd4 family HECT E3 ligases 58,59, each capturing a distinct step in the reaction. As in the case of complexes containing both E2~Ub and RING E3s, new approaches had to be devised to get around the problem of the high lability of the HECT~Ub thioester. Maspero et al.59 determined the structure of a complex that mimics the HECT~Ub thioester immediately after the transthioesterification reaction by engineering a disulfide linkage between the HECT active site cysteine and a mutated (G76C) ubiquitin C terminus. Surprisingly, the conformation of the HECT N and C lobes and the position of the donor ubiquitin are virtually superimposable with those in the HECT–UbcH5B~Ub complex57 (Fig. 4b,c). A key difference lies in the ubiquitin C terminus linked to the HECT active site cysteine: these residues are now in an extended conformation that adds a β-strand to the C-lobe β-sheet (Fig. 4d). The structure contains an additional ubiquitin that is noncovalently bound to the N lobe, potentially in a position to serve as a substrate in polyubiquitin-chain synthesis but positioned
a
Catalytic cysteine SH
C lobe
Donor ubiquitin S
E2
NH3 S
N lobe Acceptor C lobe substratebinding region
b
d
e
c
304
Acceptor substrate
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far from the E3~Ub thioester (Fig. 4d). The conformational change needed to take the C lobe with the thioester-linked donor ubiquitin to the substrate was revealed in the structure by Kamadurai and co-workers58, who used a three-way chemical cross-linker to trap a ternary HECT~Ub–substrate complex. This structure contains the yeast Nedd4 family member Rsp5 with a chemical cross-linker that couples the ubiquitin C terminus to the HECT active site cysteine as well as to a peptide substrate bound to a WW domain located N terminal to the HECT domain58 (Fig. 4e). The structure reveals a dramatic swiveling of the C lobe and the donor ubiquitin in a 130° rotation that brings the thioester in proximity to a substrate lysine (Fig. 4e). Strikingly, the donor ubiquitin is bound to the C lobe in a manner identical to that in the disulfide-linked Nedd4~Ub complex, including the β-sheet interaction of the C-terminal ubiquitin tail. The juxtaposition of N and C lobes promotes a stable intramolecular interaction that creates the active site that is viable for ubiquitin conjugation to the substrate, as predicted by combined structural and modeling studies58. Importantly, the arrangement of N and C lobes and the WW domain places spatial restrictions on the complex that favor ubiquitination of particular lysines in the bound substrate. The C terminus of HECT E3 ligases has an important role in mediating substrate specificity and catalysis, although the mechanism remains to be determined because these residues are either disordered or mediate crystal contacts in existing structures58,59. In both NEDD4L and Rsp5, a phenylalanine at the –4 or –5 position relative to the HECT domain mediates the interlobe contacts to create the active site 58,59. Substitution of alanine or leucine for phenylalanine reduced the rate of substrate ubiquitination by nearly 200-fold, and the mutant enzyme was unable to complement Rsp5 in cellular assays58. A C-terminal acidic residue in the HECT ligases NEDD4 and E6AP is required for wild-type levels of ubiquitin transfer but does not affect substrate affinity59,60. Chimeric HECT E3 ligases containing the N terminus of yeast Rsp5 and the C-terminal lobes of other HECT E3 ligases showed ubiquitin-chain linkage specificity different from that of wild-type Rsp5 (ref. 61). Similarly, the C-terminal sequence of the NEDD4Lrelated ligases influences the ubiquitin-chain linkage specificity, because substitution of the NEDD4L C terminus with the unrelated HECT E3 E6AP sequence changed the linked ubiquitin-chain product from only Lys63 (K63)-linked chains to a mixture of Lys48 (K48)- and K63-linked chains59. The mechanistic basis for these observations and the true role of the HECT C terminus remain to be resolved. Figure 4 HECT E3 ligases. (a) The HECT domain has an N lobe and C lobe, the latter of which contains the catalytic cysteine. The N lobe binds the charged E2~Ub thioester and transfers ubiquitin to the cysteine in the C lobe. The C lobe~Ub thioester rotates to bring the donor ubiquitin to the substrate-binding site, where it can be ligated to an acceptor lysine in the substrate. (b–e) Structures showing HECT N lobe (green, with surface), C lobe (green ribbons) and ubiquitin (yellow). (b) NEDD4L–UbcH5B (E2)~Ub (PDB 3JW0 (ref. 57)). (c) NEDD4~Ub with additional free ubiquitin (PDB 4BBN59). (d) Close-up view showing β-sheet interaction between the donor-ubiquitin C-terminal tail and the C lobe (PDB 4BBN59). (e) Rsp5 (HECT)~Ub–substrate (magenta) (PDB 4LCD58).
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Parkin
UBL
HHARI
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RING0
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REP
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Ariadne
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IBR RING1
IBR
RING0
Ariadne
UBA
IBR RING1 REP
active site cysteine (Fig. 5b), thus indicating that a dramatic rearrangement would be needed to bring the E2 and Parkin active Donor Ub Met1 Gly76 site cysteines together. A further component Cys885 Cys885 of autoinhibition is contributed by the REP helical linker (Fig. 5a,b), which occludes the ZF1 E2-binding site of RING1. An N-terminal His887 UBL domain, which also associates with RING2 RING1, has been shown to autoinhibit Parkin 71 , although subsequent studies report a more modest role for autoinhibition by the UBL domain as compared to RING0 (refs. 66–68). The underlying basis for the differences between the RBR-domain E3 ligases: mechanism and autoinhibition The RBR family has only recently burst upon the scene as a mecha- biochemical and structural studies is not clear, although the fact that nistically distinct class of E3s that share features of both RING and Parkin activity is affected by N-terminal tags suggests that small HECT E3s yet catalyze ubiquitination and autoregulate their activ- changes in the protein structure can affect experimental results72. ity in a distinct manner. The RBR domain was originally identified The structure of Parkin also suggests the functional basis for in Drosophila ariadne-1 (ref. 62) and in Parkin, the E3 ligase that is disease-associated Parkin mutations73, thus raising hopes for the frequently mutated in early-onset Parkinson’s disease 63. The RING- eventual development of targeted treatments for each defect66–68. between-RING name derives from the presence of two predicted HHARI65 contains the RING1, IBR and RING2 domains seen in RING domains (RING1 and RING2) separated by a conserved all three reported Parkin structures66–68 and also has the same large sequence dubbed the in-between-ring (IBR) domain62. It was thus separation between RING1 and RING2 domains (Fig. 5c). However, unexpected when the RBR proteins human homolog of ariadne instead of an N-terminal RING0 domain that blocks the RING2 active (HHARI) and Parkin were found to contain a catalytic cysteine in site, HHARI contains a C-terminal Ariadne domain that serves an RING2 that mediates ubiquitination in a HECT-like mechanism64, analogous function in masking the RING2 active site cysteine (Fig. 5c). whereas RING1 serves the canonical role of recruiting the charged E2 Despite this conserved function, the Ariadne domain bears no (refs. 64,65). Several recent structures of Parkin66–68 and HHARI65, resemblance to the inhibitory RING0 domain of Parkin. HHARI also as well as HOIP69, an RBR protein that assembles linear ubiquitin contains an N-terminal UBA domain, which in other proteins binds chains as part of the LUBAC complex70, have revealed a wealth of ubiquitin74. The three-helix UBA domain binds to another helical information on the domain organization, chemistry and regulation of region between the RING1 and IBR domains of HHARI (Fig. 5c). The these E3 ligases and have set the stage for many studies to come. presence of a UBA domain was not anticipated from the amino acid The structures of both Parkin66–68 and HHARI65 represent auto sequence; its function, including whether it binds ubiquitin, remains inhibited forms of the enzyme, because their respective domains to be determined. As in the case of Parkin, substantial conformational (Fig. 5a) are arranged in a manner that blocks E3 ligase activity rearrangements would be needed to unmask the HHARI active site (Fig. 5b,c). The modes of autoinhibition, however, differ between cysteine and bring the RING1 domain with a bound E2~Ub conthese two enzymes. Parkin contains an N-terminal ubiquitin-like jugate to the active site in RING2. These conformational changes (UBL) domain followed by a new zinc-binding domain called would involve disrupting multiple interfaces between the different RING0 and the conserved RING1, IBR and RING2 domains. Both domains and linkers in HHARI and in Parkin. An important question IBR and RING2 are zinc-binding domains with similar topologies is how the necessary conformational change is triggered, whether by that differ from the canonical cross-brace fold of RING1. The five phosphorylation (as occurs in the UBL domain of Parkin75), by other domains of Parkin associate in a compact arrangement in which post-translational modifications75, by interactions with regulatory the RING0 domain occludes the active site cysteine of RING2, thus proteins, E2 or ubiquitin, or by a combination thereof. inhibiting its activity. In agreement with this, deletion of RING0 increases autoubiquitination by Parkin 66–68. In addition, mod- Mechanism of substrate ubiquitination by RBR E3 ligases eling of E2 binding to the RING1 domain places the thioester in a Despite their general similarities to HECT E3 ligases, RBR ligases charged E2~Ub conjugate more than 50 Å away from the RING2 appear to catalyze substrate ubiquitination by a distinct mechanism.
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d
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RING2
RING2
Figure 5 RBR E3 ligase mechanism. (a) Diagram of domains in Parkin and HHARI. (b) Structure of Parkin (PDB 4K95 (ref. 67)), with domains colored as in a. RING2 active site cysteine shown as spheres. (c) Structure of HHARI with domains colored as in a and the RING2 active site cysteine shown as spheres. (d) Structure of the HOIP RING2 LDD fragment bound to donor and acceptor ubiquitin. (e) Close-up view of the HOIP active site. Arrow indicates where a thioester bond would form between the HOIP active site cysteine and the donor ubiquitin C terminus. His887 of HOIP is positioned to activate the Met1 amino group for attack on the HOIP~Ub thioester. ZF1, zinc finger 1.
Acceptor Ub
e
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RE V IE W The RBR structures revealed the presence of a conserved histidine, near the active site and important for activity65,66,68,69, as well as an aspartate and glutamate. Although the structures are reminiscent of the catalytic triad found in most deubiquitinating enzymes76, which catalyze a reaction that is essentially the reverse of ubiquitin ligation, a very recent study of the RBR ligase that assembles linear ubiquitin chains suggests a different and more compelling mechanism69. The linear ubiquitin chain–assembly complex (LUBAC) is a multiprotein RBR ligase that catalyzes peptide-bond formation between Gly76 of one ubiquitin and Met1 of the next, yielding linear ubiquitin chains70. Although the RBR domain of the HOIP subunit is sufficient to form chains, binding of HOIP to HOIL-1L and SHARPIN relieves autoinhibition and results in increased catalytic activity69,77–79. The LDD domain of HOIP (Fig. 5d) orients the acceptor ubiquitin to receive the ubiquitin from the RING2 cysteine69. A minimal fragment containing just the RING2 and LDD domains retains the ability to catalyze linear ubiquitination, albeit at low levels, thus indicating that this fragment retains the necessary determinants for linear-chain ligation69. A crystal structure of this HOIP fragment bound to ubiquitin revealed how the LDD domain forms a helical platform that binds the acceptor ubiquitin with its N-terminal methionine juxtaposed near the active site cysteine (Fig. 5d). A fortuitous crystal-packing arrangement places the C terminus of a symmetry-related ubiquitin near the active site cysteine (Fig. 5d), thus mimicking the donor ubiquitin (which in an active complex would be joined to the RING2 cysteine by a thioester linkage). Importantly, the configuration points to a role of the conserved histidine in deprotonating the N-terminal amine and activating it for attack on the E2~Ub thioester linkage (Fig. 5e). Consistently with this, the activity of a histidine-to-alanine mutant (H887A) could be partially recovered at pH 9.0, a condition favoring deprotonation of the amine. The conserved histidine presumably serves an analogous function in other RBR ligases including Parkin and HHARI, activating a lysine for attack on the thioester. Conclusions and perspective The past few years have seen much progress in elucidating the molecular mechanisms by which ubiquitin E3 ligases catalyze substrate ubiquitination. At the same time, the wealth of new structural information on all three classes of E3 ligases does as much to answer old questions as it does to pose new ones. The advances in studies of RBR ligases have been particularly remarkable, with little more than two years’ time between the discovery of their intrinsic catalytic site64 and the publication of several highly illuminating structures65–68. The next challenge will be to understand how autoinhibition is relieved, how UBL domains regulate different RBR proteins and whether the kinds of large domain motions seen in HECT E3 ligases also accompany the two-step reaction mechanisms of RBR E3s. Structures of complexes with E2 enzymes will be very important for further understanding of these reaction steps. A full understanding of linear ubiquitination by LUBAC will also depend on determining how the other subunits, HOIL-1L and SHARPIN, stimulate the activity of HOIP. Because HOIL-1L also contains an RBR domain, it will be interesting to see whether there is any coordination between HOIL-1L and HOIP RBR domains. An ultimate understanding of two-step ubiquitin ligation by both RBR and HECT E3s will probably require additional structures that capture all major steps from transthioesterification to substrate ubiquitination. There is more to be learned about how E3 ligases target specific substrate lysines or protein N termini for modification. Although the substrate specificity of ubiquitination is understood at a mechanistic level in a handful of cases, these are the exceptions rather than the rule. In the case of polyubiquitin-chain assembly, the last thioester in 306
the ubiquitination cascade determines chain linkage type: thus, the E2 largely governs chain linkage in the case of polyubiquitination catalyzed by RING E3s, whereas HECT and RBR E3 ligases govern polyubiquitin-chain type. The recent study of HOIP explains its specificity for linear ubiquitination69, but a molecular explanation of the chain linkage specificity of Parkin and other RBR or HECT E3s is still lacking. E2 linkage specificity is best understood for Ubc13–Mms2, in which the Mms2 subunit positions the acceptor ubiquitin to promote conjugation to K63 of ubiquitin23. For substrates other than ubiquitin, there is better understanding of how a wide variety of substrates are recruited by different E3 ligases10 but less understanding of how particular lysines are targeted within regions of the protein that are within striking distance of the ubiquitin thioester. Structures such as that of the HECT E3 Rsp5 bound to both ubiquitin and substrate58 are a start and will, we hope, be followed by multiple examples from all classes of E3 ligases. These structures are sure to reveal more about the role of E3 ligase complex formation on catalytic activity, substrate binding and regulation, thus revealing the inner workings of the ubiquitin-conjugation pathway. Acknowledgments We thank R. Hay, A. Plechanovová and B. Schulman for providing coordinates of modeled complexes. C.W. acknowledges grant support from the US National Institutes of Health (GM095822) and National Science Foundation (MCB0920082). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006). 2. Ulrich, H.D. & Walden, H. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489 (2010). 3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012). 4. Pickart, C.M. & Eddins, M.J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004). 5. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell Biol. 11, 123–132 (2009). 6. Scaglione, K.M. et al. The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates. J. Biol. Chem. 288, 18784–18788 (2013). 7. Tatham, M.H., Plechanovová, A., Jaffray, E.G., Salmen, H. & Hay, R.T. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini. Biochem. J. 453, 137–145 (2013). 8. Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009). 9. Li, W. et al. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS ONE 3, e1487 (2008). 10. Metzger, M.B., Hristova, V.A. & Weissman, A.M. HECT and RING finger families of E3 ubiquitin ligases at a glance. J. Cell Sci. 125, 531–537 (2012). 11. Budhidarmo, R., Nakatani, Y. & Day, C.L. RINGs hold the key to ubiquitin transfer. Trends Biochem. Sci. 37, 58–65 (2012). 12. Huibregtse, J.M., Scheffner, M., Beaudenon, S. & Howley, P.M. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92, 2563–2567 (1995). 13. Wenzel, D.M. & Klevit, R.E. Following Ariadne’s thread: a new perspective on RBR ubiquitin ligases. BMC Biol. 10, 24 (2012). 14. van Wijk, S.J. & Timmers, H.T. The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J. 24, 981–993 (2010). 15. Wenzel, D.M., Stoll, K.E. & Klevit, R.E. E2s: structurally economical and functionally replete. Biochem. J. 433, 31–42 (2011). 16. Wu, P.Y. et al. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 22, 5241–5250 (2003). 17. Berndsen, C.E., Wiener, R., Yu, I.W., Ringel, A.E. & Wolberger, C. A conserved asparagine has a structural role in ubiquitin-conjugating enzymes. Nat. Chem. Biol. 9, 154–156 (2013). 18. Yunus, A.A. & Lima, C.D. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat. Struct. Mol. Biol. 13, 491–499 (2006). 19. 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).
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focus on ubiquitin
New insights into ubiquitin E3 ligase mechanism Christopher E Berndsen1 & Cynthia Wolberger2,3
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E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated. The attachment of the small protein ubiquitin to substrates regulates a vast array of processes in eukaryotes1,2. Substrates can be modified with a single ubiquitin (Ub) or with a polyubiquitin chain in which one ubiquitin is conjugated to the next3. Ubiquitin is covalently attached to substrate lysines in a three-enzyme cascade catalyzed by E1, E2 and E3 enzymes4, thus resulting in an isopeptide linkage between the ubiquitin C terminus and the ε-amino group of lysine (Box 1). Ubiquitin itself can be ubiquitinated at any one of its seven lysines to give rise to different types of polyubiquitin chains3. Recent studies have shown that ubiquitin can also be covalently linked to N-terminal α-amino groups via a peptide linkage to yield linear polyubiquitin chains5 or N-terminal ubiquitin fusions6,7. Ubiquitin is activated in an ATP-dependent reaction by the E1 enzyme, which forms a thioester with the ubiquitin C terminus, and is then transferred to the active site cysteine of the E2 enzyme to yield an E2~Ub thioester inter mediate (Box 1). The E3 ligase binds to both the E2~Ub thioester and the substrate, catalyzing transfer of the ubiquitin from the active site cysteine of the E2 to the substrate lysine or N terminus8. E3 ligases thus have dual roles as both molecular matchmaker and catalyst, bringing together the right E2 with the right substrate and greatly increasing the rate of ubiquitin transfer. The need to specifically target a broad array of substrates accounts for the great diversity among the estimated >600 human E3s9. Nonetheless, E3 ligases fall into three classes10, each characterized by conserved structural domains and the mechanism by which ubiquitin is transferred from the E2 to the substrate. The really interesting new gene (RING) family catalyzes direct transfer of ubiquitin from the E2 enzyme to the substrate, simultaneously binding both the E2~Ub thioester and the substrate8,11. In contrast, the homology to E6AP C terminus (HECT) and the RING-between-RING (RBR) family E3s ubiquitinate substrates in a two-step reaction in which ubiquitin is transferred from the E2 to an active site cysteine in the E3 and then from the E3 to the substrate12,13. Despite numerous structural 1Department
of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, USA. 2Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 3Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Correspondence should be addressed to C.W. ([email protected]). Received 21 November 2013; accepted 28 January 2014; published online 4 April 2014; doi:10.1038/nsmb.2780
and biochemical studies of ubiquitin conjugation by both E2 and E3 enzymes, the catalytic mechanisms by which E3 ligases enhance rates of ubiquitin transfer have remained remarkably elusive. The mystery of the E3 ligase catalytic mechanism has finally yielded to a series of recent structural and biochemical studies that have given insights into the mechanism by which all three classes of E3 ligases stimulate substrate ubiquitination as well as the way in which E3 ligase activity is regulated. E2 enzymes, essential partners of E3s All E3 ligases bind an E2~Ub thioester and either catalyze transfer of ubiquitin from the E2 to a substrate lysine via an aminolysis reaction, as is the case for RING E3s, or to another cysteine in the E3 via a transthioesterification reaction, as is the case for HECT and RBR E3s. The RING E3 ligases contain no active site residues per se, but they instead greatly enhance the rate of ubiquitin transfer from the E2 active site to the substrate lysine (or N terminus). In this reaction, the amino group in the substrate attacks the thioester linkage between the ubiquitin C terminus and the active site cysteine of the E2, thus leading to hydrolysis of the thioester and formation of an isopeptide (or peptide) bond with the substrate. E2 enzymes contain a highly conserved catalytic domain of ~150 amino acids 14,15. E2 enzymes contain, in addition to the active site cysteine, a conserved asparagine that had been proposed to stabilize an oxyanion reaction intermediate16 but appears to have an essential role in stabilizing the E2 structure in the vicinity of the active site 17. Although there are no other highly conserved residues in the region of the E2 active site, there are typically one or more acidic residues in the vicinity of the active site cysteine that have been proposed to facilitate ubiquitin transfer by orienting and deprotonating the attacking lysine18,19. In the E2 Cdc34, which lacks the corresponding acidic residues, a substrate glutamic acid neighboring the acceptor lysine has been proposed to have an analogous role20,21. In the absence of an E3, the ubiquitin in the E2~Ub thioester is highly mobile, with NMR and crystallographic studies showing multiple conformations of ubiquitin relative to the E2 (refs. 22–25). An important question for understanding all types of E3 ligases was therefore how and whether the donor ubiquitin was positioned for the next step in ubiquitin transfer and how the substrate lysine (in the case of RING E3s) or cysteine (in the case of HECT and RBR E3s) was oriented to attack the E2~Ub thioester linkage.
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Box 1 The ubiquitin cascade and chain formation
© 2014 Nature America, Inc. All rights reserved.
(a) The ubiquitination cascade begins with ATP-dependent charging of the E1 enzyme and results in formation of a thioester bond between the ubiquitin C terminus and the E1 active site cysteine. Ubiquitin is transferred to the E2 active site cysteine in a transthioesterification reaction. An E3 ligase catalyzes transfer of the ubiquitin from the active site cysteine of the E2 to a primary amine on a lysine side chain or protein N terminus. There are three classes of E3 ligases: RING E3s, which bind to both E2~Ub thioester and substrate and catalyze attack of the substrate lysine on the thioester, and HECT and RBR E3s, which both have active site cysteines and catalyze substrate ubiquitination in a two-step reaction involving formation of a thioester with the HECT or RBR E3 followed by attack of the substrate lysine or N terminus on the E3~Ub thioester to form an isopeptide (or peptide) linkage between the ubiquitin C terminus and lysine (or the protein N terminus). (b) Polyubiquitin chains. Ubiquitin (PDB 1UBQ80) has seven lysines and an amino terminus (Met1) that can be ubiquitinated to give rise to different types of polyubiquitin chains. Diubiquitin structures are shown for linear (Met1-linked) (PDB 2W9N81), K63-linked (from PDB 3HM3 (ref. 82)) and K48-linked diubiquitin (1AAR83).
a
ATP
AMP
Ub
SH
S
E1
SH
E1
E1 Ub
Ub SH
S
E2
E2 HECT RBR
RING
Ub Lys
S
E2
Substrate
SH
Ub
E3
Lys
S E2
Substrate
Ub
E3
npg
SH E2
S
Ub
Lys
Ub
Ub
Substrate Ub
E3
Lys
SH E2
Substrate
Ub Lys
SH E2
SH
E3
Substrate
E3
b K63 Met1
K33
K6
K48
K29
K27
K63
Met1
K48
K11
C
Ubiquitin
302
Linear diubiquitin
K63-linked diubiquitin
K48-linked diubiquitin
Catalysis of ubiquitin transfer by RING E3s RING E3 ligases contain a RING (or RING-like) domain that is responsible for both binding to the E2 and stimulating ubiquitin transfer26. The RING domain adopts a conserved cross-brace structure with two structural Zn2+ ions (Fig. 1a) and can be monomeric27–30 or dimeric25,26,31,32, with dimerization mediated either by the RING domain alone or facilitated by flanking helical regions32–34 (Fig. 1b). A related domain known as the U box has a similar function and fold but has a hydrophobic core in place of the structural metal ions35. RING domains bind to the N-terminal helix of E2 conjugating enzymes30 (Fig. 1c). In the presence or absence of an E2-bound ubiquitin, the E2-E3 interaction is transient and appears to compete with binding of the E1 enzyme to E2 (ref. 36). In addition to the E2-binding domain, RING E3 ligases contain multiple additional domains including those that recruit the substrate for ubiquitin conjugation (reviewed in ref. 8). A subset of RING E3 ligases known as cullin E3s (Fig. 1d) are multisubunit proteins containing a scaffold protein, called the cullin protein, a RING-box protein, containing a domain similar to the RING domain found in single-polypeptide E3 ligases, and a bridging protein, called an F-box protein, for binding the substrate (reviewed in refs. 37,38). Whereas the determinants of RING domain–E2 interactions have been elucidated through studies of many RING domain–E2 complexes27,29,30,34,39,40, these structures provided little insight into the mechanism by which RING domains stimulate ubiquitin transfer. The distance between the RING domain and the E2 active site (Fig. 1c) ruled out direct participation of RING residues in catalysis, and proposals that RING binding communicated allosteric changes to the active site lacked support41. Studies showed that RING domains bind more tightly to E2~Ub conjugates17,42–44, pointing to a role for RING domains in contacting the donor ubiquitin. However, a confounding obstacle was the inability to trap a complex between a RING domain and an E2~Ub thioester for structural studies, because addition of the RING causes rapid hydrolysis, even when a more stable oxyester mimic is used19. This barrier was finally overcome by a clever replacement of the native thioester with an isopeptide linkage, which was done by substituting the active site cysteine of the E2 UbcH5A with a lysine and charging the mutant enzyme with ubi quitin19. In the structure of this E2~Ub conjugate bound to the RING domain of RNF4, Plechanovová et al.19 showed that the RING domain contacts a hydrophobic patch on the donor ubiquitin; this interaction immobilizes the donor ubiquitin and positions its C terminus in a structurally conserved groove in the E2 (Fig. 2a,b). The position of the ubiquitin tail in the RNF4–UbcH5 structure resembles a product complex between the ubiquitin-like protein SUMO and its E2 (ref. 45). In an independent approach used to determine the structure of the BIRC7 RING domain bound to UbcH5B~Ub28, two mutations were used to stabilize the E2~Ub linkage: the active site cysteine was mutated to serine, which forms a more stable ester linkage with ubiquitin, and the conserved asparagine was mutated to alanine (N77A). As with RNF4, both subunits of the dimeric BIRC7 RING domain position the donor ubiquitin attached to UbcH5B. Both of these studies agree with solution NMR experiments showing that the RING heterodimer, BRCA1–BARD1, and the monomeric U box, E4B, restrict the position of the donor ubiquitin on the E2 (ref. 46). Whether this mechanism of conformational selection (Fig. 2c) applies to RING proteins found in the cullin family of ligases has not been demonstrated but is likely, given the similarities in the E2 binding modes. How does conformational selection increase the rate of ubiquitin transfer? Positioning ubiquitin presumably places the reactive thioester between E2 and the ubiquitin C terminus in the correct
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orientation to react with the attacking lysine (Fig. 2d). Immobilizing a bond can increase the rate of reaction47,48 and consequently also account for the increased rate of thioester hydrolysis in the presence of a RING domain49. Additional contributions may come from the correct positioning of the thioester and acceptor lysine in relation to conserved residues that flank the immobilized ubiquitin tail and the active site cysteine (Fig. 2b). These include residues critical for activity, such as acidic residues (Asp117 in UbcH5B) that have been proposed to activate the attacking lysine17,19,28,45, as well as a conserved asparagine (Asn77 in UbcH5B) that has been proposed to stabilize an oxyanion intermediate16 but which was recently shown to have an important structural role in configuring residues in the vicinity of the active site17 (Fig. 2d). Further studies will be needed to elucidate the mechanism by which these residues contribute to catalysis once the donor ubiquitin has been correctly positioned on the E2. Although the RING domain has the primary role in catalyzing ubiquitin transfer, recent studies have shown that some RING E3 ligases contain additional domains that bind to the E2 and influence substrate ubiquitination. The RING E3 gp78 contains an additional helical domain, known as the G2BR50, that binds to the E2 β-sheet on the face opposite that of the active site (Fig. 3a), known as the ‘back side’, to which free ubiquitin has been observed to bind in solution51. G2BR binding is proposed to trigger allosteric changes in the E2 Ube2g2, opening the active site for substrate binding and stimulating ubiquitination50. More recent work shows that RING-domain binding to Ube2g2 antagonizes G2BR binding, promoting E2 enzyme exchange during processive ubiquitination by gp78 (ref. 52). Rad18, the E3 ligase that catalyzes monoubiquitination of PCNA, contains an N-terminal RING domain that binds to the E2 Rad6, as well as a distinct region at the C terminus of the protein53 that contacts
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Figure 1 RING E3 ligases. Bound zinc atoms in all structures are depicted as red spheres. (a) Monomeric RING from c-CBL (PDB 1FBV30). (b) RING homodimer from BIRC7 (PDB 4AUQ28). Two α-helices contribute additional dimer contacts. (c) TRAF6 ring bound to the E2 Ubc13 (PDB 3HCT29). The active site cysteine of Ubc13 is depicted as spheres. (d) Cullin RING E3 ligases are multisubunit proteins with separate substrate-binding domains (the F box protein (Fbp)) and RING, which binds the E2. Composite model assembled from PDB 1P22 (ref. 84), 1LDJ85, 3DQV86 and 4AP4 (ref. 19). CTD, C-terminal domain; NTD, N-terminal domain.
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the E2 back side (Fig. 3b) and suppresses the intrinsic polyubiquitination activity of Rad6 (ref. 39). The Cue1 subunit of yeast ERAD E3 ligase complexes contains a U7BR domain that also binds to the back side of Ubc7 (Fig. 3c) and stimulates both E1 charging and substrate ubiquitination54. Whereas the additional domains in all three of these examples all contact the same surface of the E2, neither the structures nor the mode of interaction is conserved, thus making it difficult to derive any general principles for how these proteins stimulate E2 activity. Understanding the underlying mechanism by which these additional interactions with the E2 contribute to substrate ubiquitination will require determining the somewhat mysterious role of noncovalent ubiquitin binding to the E2 back side51. Two-step ubiquitination by HECT E3 ligases In contrast to RING E3 ligases, which catalyze direct attack of the substrate lysine on the E2~Ub thioester, HECT-domain E3 ligases catalyze two distinct reactions (Box 1): a transthioesterification reaction, in which ubiquitin is transferred from the E2 active site cysteine to a cysteine in the HECT domain, and a subsequent attack on the HECT~Ub thioester by a substrate lysine12. The conserved HECT domain itself comprises an N lobe that binds to the E2 and a Figure 2 Mechanism by which RING E3s stimulate ubiquitin transfer. (a) Structure of RNF4 RING dimer (green, light green) bound to the E2 UbcH5A (blue) charged with ubiquitin (yellow) (PDB 4AP4 (ref. 19)). Omitted from this view is a second E2~Ub conjugate that binds to the light-green RING monomer. (b) Close-up view showing hydrogen-bond interactions between the C-terminal tail of ubiquitin (yellow), E2 (blue) and an arginine in the RNF4 RING (green). The E2 active site cysteine is replaced with a lysine (C85K), which forms an isopeptide bond with the ubiquitin C terminus in the crystallized complex. (c) Activation of E2~Ub for ubiquitin transfer by conformational selection. The thioester-linked ubiquitin is free to adopt multiple positions, but binding of the RING to E2 immobilizes the donor ubiquitin. (d) Model for attack of acceptor lysine (magenta) on the thioester linkage. Asp117 has been proposed to deprotonate the lysine and orient it for nucleophilic attack. Asn77 is in van der Waals contact with the modeled thioester and also forms two conserved hydrogen bonds with backbone atoms.
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Figure 3 E2 back-side binding by additional domains in RING E3 ligases. (a) The E2 Ube2g2 (blue) bound to the G2BR (magenta) and RING (green) of the E3 ligase gp78 (PDB 4LAD52). (b) The Rad6-binding domain (R6BD, magenta) of Rad18 bound to the E2 Rad6 (blue) (PDB 2YBF39). (c) The U7BR (magenta) domain of Cue1 bound to the E2 Ubc7 (blue) (PDB 4JQU54).
b
C lobe that contains the active site cysteine (Fig. 4a). A flexible tether between the N and C lobes permits multiple relative configurations of these two lobes that have proved critical to HECT E3 activity (Fig. 4a). Early structural studies of the E6AP55 and WWP1 (ref. 56) HECT-domain proteins showed the N lobe and C lobe to be distant from each another and suggested that a conformational rearrangement of the N and C lobes was needed to bring the E2~Ub thioester sufficiently close to the active site cysteine in the HECT C lobe. A structure of the HECT E3 NEDD4L bound to a UbcH5B~Ub conjugate57 showed that the C lobe rotated markedly, thus causing the HECT domain to adopt a more compact conformation that brought the E2~Ub thioester near the cysteine in the C lobe and permitted direct contacts between the N lobe and the ubiquitin (Fig. 4b). Although the active site cysteine of NEDD4L is still 8 Å from the cysteine of UbcH5B in this structure57, a modest additional rotation of the C lobe closes the gap sufficiently to permit the transthioesterification reaction to occur. The remaining question of how a charged HECT E3~Ub thioester transfers ubiquitin to a substrate lysine was addressed only recently by two structural studies of Nedd4 family HECT E3 ligases 58,59, each capturing a distinct step in the reaction. As in the case of complexes containing both E2~Ub and RING E3s, new approaches had to be devised to get around the problem of the high lability of the HECT~Ub thioester. Maspero et al.59 determined the structure of a complex that mimics the HECT~Ub thioester immediately after the transthioesterification reaction by engineering a disulfide linkage between the HECT active site cysteine and a mutated (G76C) ubiquitin C terminus. Surprisingly, the conformation of the HECT N and C lobes and the position of the donor ubiquitin are virtually superimposable with those in the HECT–UbcH5B~Ub complex57 (Fig. 4b,c). A key difference lies in the ubiquitin C terminus linked to the HECT active site cysteine: these residues are now in an extended conformation that adds a β-strand to the C-lobe β-sheet (Fig. 4d). The structure contains an additional ubiquitin that is noncovalently bound to the N lobe, potentially in a position to serve as a substrate in polyubiquitin-chain synthesis but positioned
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far from the E3~Ub thioester (Fig. 4d). The conformational change needed to take the C lobe with the thioester-linked donor ubiquitin to the substrate was revealed in the structure by Kamadurai and co-workers58, who used a three-way chemical cross-linker to trap a ternary HECT~Ub–substrate complex. This structure contains the yeast Nedd4 family member Rsp5 with a chemical cross-linker that couples the ubiquitin C terminus to the HECT active site cysteine as well as to a peptide substrate bound to a WW domain located N terminal to the HECT domain58 (Fig. 4e). The structure reveals a dramatic swiveling of the C lobe and the donor ubiquitin in a 130° rotation that brings the thioester in proximity to a substrate lysine (Fig. 4e). Strikingly, the donor ubiquitin is bound to the C lobe in a manner identical to that in the disulfide-linked Nedd4~Ub complex, including the β-sheet interaction of the C-terminal ubiquitin tail. The juxtaposition of N and C lobes promotes a stable intramolecular interaction that creates the active site that is viable for ubiquitin conjugation to the substrate, as predicted by combined structural and modeling studies58. Importantly, the arrangement of N and C lobes and the WW domain places spatial restrictions on the complex that favor ubiquitination of particular lysines in the bound substrate. The C terminus of HECT E3 ligases has an important role in mediating substrate specificity and catalysis, although the mechanism remains to be determined because these residues are either disordered or mediate crystal contacts in existing structures58,59. In both NEDD4L and Rsp5, a phenylalanine at the –4 or –5 position relative to the HECT domain mediates the interlobe contacts to create the active site 58,59. Substitution of alanine or leucine for phenylalanine reduced the rate of substrate ubiquitination by nearly 200-fold, and the mutant enzyme was unable to complement Rsp5 in cellular assays58. A C-terminal acidic residue in the HECT ligases NEDD4 and E6AP is required for wild-type levels of ubiquitin transfer but does not affect substrate affinity59,60. Chimeric HECT E3 ligases containing the N terminus of yeast Rsp5 and the C-terminal lobes of other HECT E3 ligases showed ubiquitin-chain linkage specificity different from that of wild-type Rsp5 (ref. 61). Similarly, the C-terminal sequence of the NEDD4Lrelated ligases influences the ubiquitin-chain linkage specificity, because substitution of the NEDD4L C terminus with the unrelated HECT E3 E6AP sequence changed the linked ubiquitin-chain product from only Lys63 (K63)-linked chains to a mixture of Lys48 (K48)- and K63-linked chains59. The mechanistic basis for these observations and the true role of the HECT C terminus remain to be resolved. Figure 4 HECT E3 ligases. (a) The HECT domain has an N lobe and C lobe, the latter of which contains the catalytic cysteine. The N lobe binds the charged E2~Ub thioester and transfers ubiquitin to the cysteine in the C lobe. The C lobe~Ub thioester rotates to bring the donor ubiquitin to the substrate-binding site, where it can be ligated to an acceptor lysine in the substrate. (b–e) Structures showing HECT N lobe (green, with surface), C lobe (green ribbons) and ubiquitin (yellow). (b) NEDD4L–UbcH5B (E2)~Ub (PDB 3JW0 (ref. 57)). (c) NEDD4~Ub with additional free ubiquitin (PDB 4BBN59). (d) Close-up view showing β-sheet interaction between the donor-ubiquitin C-terminal tail and the C lobe (PDB 4BBN59). (e) Rsp5 (HECT)~Ub–substrate (magenta) (PDB 4LCD58).
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active site cysteine (Fig. 5b), thus indicating that a dramatic rearrangement would be needed to bring the E2 and Parkin active Donor Ub Met1 Gly76 site cysteines together. A further component Cys885 Cys885 of autoinhibition is contributed by the REP helical linker (Fig. 5a,b), which occludes the ZF1 E2-binding site of RING1. An N-terminal His887 UBL domain, which also associates with RING2 RING1, has been shown to autoinhibit Parkin 71 , although subsequent studies report a more modest role for autoinhibition by the UBL domain as compared to RING0 (refs. 66–68). The underlying basis for the differences between the RBR-domain E3 ligases: mechanism and autoinhibition The RBR family has only recently burst upon the scene as a mecha- biochemical and structural studies is not clear, although the fact that nistically distinct class of E3s that share features of both RING and Parkin activity is affected by N-terminal tags suggests that small HECT E3s yet catalyze ubiquitination and autoregulate their activ- changes in the protein structure can affect experimental results72. ity in a distinct manner. The RBR domain was originally identified The structure of Parkin also suggests the functional basis for in Drosophila ariadne-1 (ref. 62) and in Parkin, the E3 ligase that is disease-associated Parkin mutations73, thus raising hopes for the frequently mutated in early-onset Parkinson’s disease 63. The RING- eventual development of targeted treatments for each defect66–68. between-RING name derives from the presence of two predicted HHARI65 contains the RING1, IBR and RING2 domains seen in RING domains (RING1 and RING2) separated by a conserved all three reported Parkin structures66–68 and also has the same large sequence dubbed the in-between-ring (IBR) domain62. It was thus separation between RING1 and RING2 domains (Fig. 5c). However, unexpected when the RBR proteins human homolog of ariadne instead of an N-terminal RING0 domain that blocks the RING2 active (HHARI) and Parkin were found to contain a catalytic cysteine in site, HHARI contains a C-terminal Ariadne domain that serves an RING2 that mediates ubiquitination in a HECT-like mechanism64, analogous function in masking the RING2 active site cysteine (Fig. 5c). whereas RING1 serves the canonical role of recruiting the charged E2 Despite this conserved function, the Ariadne domain bears no (refs. 64,65). Several recent structures of Parkin66–68 and HHARI65, resemblance to the inhibitory RING0 domain of Parkin. HHARI also as well as HOIP69, an RBR protein that assembles linear ubiquitin contains an N-terminal UBA domain, which in other proteins binds chains as part of the LUBAC complex70, have revealed a wealth of ubiquitin74. The three-helix UBA domain binds to another helical information on the domain organization, chemistry and regulation of region between the RING1 and IBR domains of HHARI (Fig. 5c). The these E3 ligases and have set the stage for many studies to come. presence of a UBA domain was not anticipated from the amino acid The structures of both Parkin66–68 and HHARI65 represent auto sequence; its function, including whether it binds ubiquitin, remains inhibited forms of the enzyme, because their respective domains to be determined. As in the case of Parkin, substantial conformational (Fig. 5a) are arranged in a manner that blocks E3 ligase activity rearrangements would be needed to unmask the HHARI active site (Fig. 5b,c). The modes of autoinhibition, however, differ between cysteine and bring the RING1 domain with a bound E2~Ub conthese two enzymes. Parkin contains an N-terminal ubiquitin-like jugate to the active site in RING2. These conformational changes (UBL) domain followed by a new zinc-binding domain called would involve disrupting multiple interfaces between the different RING0 and the conserved RING1, IBR and RING2 domains. Both domains and linkers in HHARI and in Parkin. An important question IBR and RING2 are zinc-binding domains with similar topologies is how the necessary conformational change is triggered, whether by that differ from the canonical cross-brace fold of RING1. The five phosphorylation (as occurs in the UBL domain of Parkin75), by other domains of Parkin associate in a compact arrangement in which post-translational modifications75, by interactions with regulatory the RING0 domain occludes the active site cysteine of RING2, thus proteins, E2 or ubiquitin, or by a combination thereof. inhibiting its activity. In agreement with this, deletion of RING0 increases autoubiquitination by Parkin 66–68. In addition, mod- Mechanism of substrate ubiquitination by RBR E3 ligases eling of E2 binding to the RING1 domain places the thioester in a Despite their general similarities to HECT E3 ligases, RBR ligases charged E2~Ub conjugate more than 50 Å away from the RING2 appear to catalyze substrate ubiquitination by a distinct mechanism.
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d
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Figure 5 RBR E3 ligase mechanism. (a) Diagram of domains in Parkin and HHARI. (b) Structure of Parkin (PDB 4K95 (ref. 67)), with domains colored as in a. RING2 active site cysteine shown as spheres. (c) Structure of HHARI with domains colored as in a and the RING2 active site cysteine shown as spheres. (d) Structure of the HOIP RING2 LDD fragment bound to donor and acceptor ubiquitin. (e) Close-up view of the HOIP active site. Arrow indicates where a thioester bond would form between the HOIP active site cysteine and the donor ubiquitin C terminus. His887 of HOIP is positioned to activate the Met1 amino group for attack on the HOIP~Ub thioester. ZF1, zinc finger 1.
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RE V IE W The RBR structures revealed the presence of a conserved histidine, near the active site and important for activity65,66,68,69, as well as an aspartate and glutamate. Although the structures are reminiscent of the catalytic triad found in most deubiquitinating enzymes76, which catalyze a reaction that is essentially the reverse of ubiquitin ligation, a very recent study of the RBR ligase that assembles linear ubiquitin chains suggests a different and more compelling mechanism69. The linear ubiquitin chain–assembly complex (LUBAC) is a multiprotein RBR ligase that catalyzes peptide-bond formation between Gly76 of one ubiquitin and Met1 of the next, yielding linear ubiquitin chains70. Although the RBR domain of the HOIP subunit is sufficient to form chains, binding of HOIP to HOIL-1L and SHARPIN relieves autoinhibition and results in increased catalytic activity69,77–79. The LDD domain of HOIP (Fig. 5d) orients the acceptor ubiquitin to receive the ubiquitin from the RING2 cysteine69. A minimal fragment containing just the RING2 and LDD domains retains the ability to catalyze linear ubiquitination, albeit at low levels, thus indicating that this fragment retains the necessary determinants for linear-chain ligation69. A crystal structure of this HOIP fragment bound to ubiquitin revealed how the LDD domain forms a helical platform that binds the acceptor ubiquitin with its N-terminal methionine juxtaposed near the active site cysteine (Fig. 5d). A fortuitous crystal-packing arrangement places the C terminus of a symmetry-related ubiquitin near the active site cysteine (Fig. 5d), thus mimicking the donor ubiquitin (which in an active complex would be joined to the RING2 cysteine by a thioester linkage). Importantly, the configuration points to a role of the conserved histidine in deprotonating the N-terminal amine and activating it for attack on the E2~Ub thioester linkage (Fig. 5e). Consistently with this, the activity of a histidine-to-alanine mutant (H887A) could be partially recovered at pH 9.0, a condition favoring deprotonation of the amine. The conserved histidine presumably serves an analogous function in other RBR ligases including Parkin and HHARI, activating a lysine for attack on the thioester. Conclusions and perspective The past few years have seen much progress in elucidating the molecular mechanisms by which ubiquitin E3 ligases catalyze substrate ubiquitination. At the same time, the wealth of new structural information on all three classes of E3 ligases does as much to answer old questions as it does to pose new ones. The advances in studies of RBR ligases have been particularly remarkable, with little more than two years’ time between the discovery of their intrinsic catalytic site64 and the publication of several highly illuminating structures65–68. The next challenge will be to understand how autoinhibition is relieved, how UBL domains regulate different RBR proteins and whether the kinds of large domain motions seen in HECT E3 ligases also accompany the two-step reaction mechanisms of RBR E3s. Structures of complexes with E2 enzymes will be very important for further understanding of these reaction steps. A full understanding of linear ubiquitination by LUBAC will also depend on determining how the other subunits, HOIL-1L and SHARPIN, stimulate the activity of HOIP. Because HOIL-1L also contains an RBR domain, it will be interesting to see whether there is any coordination between HOIL-1L and HOIP RBR domains. An ultimate understanding of two-step ubiquitin ligation by both RBR and HECT E3s will probably require additional structures that capture all major steps from transthioesterification to substrate ubiquitination. There is more to be learned about how E3 ligases target specific substrate lysines or protein N termini for modification. Although the substrate specificity of ubiquitination is understood at a mechanistic level in a handful of cases, these are the exceptions rather than the rule. In the case of polyubiquitin-chain assembly, the last thioester in 306
the ubiquitination cascade determines chain linkage type: thus, the E2 largely governs chain linkage in the case of polyubiquitination catalyzed by RING E3s, whereas HECT and RBR E3 ligases govern polyubiquitin-chain type. The recent study of HOIP explains its specificity for linear ubiquitination69, but a molecular explanation of the chain linkage specificity of Parkin and other RBR or HECT E3s is still lacking. E2 linkage specificity is best understood for Ubc13–Mms2, in which the Mms2 subunit positions the acceptor ubiquitin to promote conjugation to K63 of ubiquitin23. For substrates other than ubiquitin, there is better understanding of how a wide variety of substrates are recruited by different E3 ligases10 but less understanding of how particular lysines are targeted within regions of the protein that are within striking distance of the ubiquitin thioester. Structures such as that of the HECT E3 Rsp5 bound to both ubiquitin and substrate58 are a start and will, we hope, be followed by multiple examples from all classes of E3 ligases. These structures are sure to reveal more about the role of E3 ligase complex formation on catalytic activity, substrate binding and regulation, thus revealing the inner workings of the ubiquitin-conjugation pathway. Acknowledgments We thank R. Hay, A. Plechanovová and B. Schulman for providing coordinates of modeled complexes. C.W. acknowledges grant support from the US National Institutes of Health (GM095822) and National Science Foundation (MCB0920082). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006). 2. Ulrich, H.D. & Walden, H. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489 (2010). 3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012). 4. Pickart, C.M. & Eddins, M.J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004). 5. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell Biol. 11, 123–132 (2009). 6. Scaglione, K.M. et al. The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates. J. Biol. Chem. 288, 18784–18788 (2013). 7. Tatham, M.H., Plechanovová, A., Jaffray, E.G., Salmen, H. & Hay, R.T. Ube2W conjugates ubiquitin to α-amino groups of protein N-termini. Biochem. J. 453, 137–145 (2013). 8. Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009). 9. Li, W. et al. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle’s dynamics and signaling. PLoS ONE 3, e1487 (2008). 10. Metzger, M.B., Hristova, V.A. & Weissman, A.M. HECT and RING finger families of E3 ubiquitin ligases at a glance. J. Cell Sci. 125, 531–537 (2012). 11. Budhidarmo, R., Nakatani, Y. & Day, C.L. RINGs hold the key to ubiquitin transfer. Trends Biochem. Sci. 37, 58–65 (2012). 12. Huibregtse, J.M., Scheffner, M., Beaudenon, S. & Howley, P.M. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92, 2563–2567 (1995). 13. Wenzel, D.M. & Klevit, R.E. Following Ariadne’s thread: a new perspective on RBR ubiquitin ligases. BMC Biol. 10, 24 (2012). 14. van Wijk, S.J. & Timmers, H.T. The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J. 24, 981–993 (2010). 15. Wenzel, D.M., Stoll, K.E. & Klevit, R.E. E2s: structurally economical and functionally replete. Biochem. J. 433, 31–42 (2011). 16. Wu, P.Y. et al. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 22, 5241–5250 (2003). 17. Berndsen, C.E., Wiener, R., Yu, I.W., Ringel, A.E. & Wolberger, C. A conserved asparagine has a structural role in ubiquitin-conjugating enzymes. Nat. Chem. Biol. 9, 154–156 (2013). 18. Yunus, A.A. & Lima, C.D. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat. Struct. Mol. Biol. 13, 491–499 (2006). 19. 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).
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