E3 Rad18 promotes monoubiquitination rather than chain formation by E2 Rad6

Richard G. Hibberta,1, Anding Huangb,1, Rolf Boelensb,2, and Titia K. Sixmaa,2

aDivision of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; and bDepartment of Nuclear Magnetic Resonance Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved February 18, 2011 (received for review November 24, 2010)

In ubiquitin conjugation, different combinations of E2 and E3 interaction with ubiquitin (14), and a similar small ubiquitin-like catalyse either monoubiquitination or ubiquitin chain modifier (SUMO) interaction is required for the E2 enzyme formation. The E2/E3 complex Rad6/Rad18 exclusively monoubi- Ubc9 (15–17). Both of these interactions are remarkably similar quitinates the proliferating cell nuclear antigen (PCNA) to signal to the ubiquitin interaction of MMS2 because they occur on the for “error prone” DNA damage tolerance, whereas a different set “backside” of the enzymes, distant from the active site cysteine. of conjugation enzymes is required for ubiquitin chain formation Loops close to the active site cysteine are important in the chain- on PCNA. Here we show that human E2 enzyme Rad6b is intrinsi- formation activity of Ubc1 (18) and Cdc34 (4), whereas Ube2C, cally capable of catalyzing ubiquitin chain formation. This activity is together with the APC/C, recognizes TEK boxes found both on prevented during PCNA ubiquitination by the interaction of Rad6 the target securin and close to K11 on ubiquitin (19). It remains with E3 enzyme Rad18. Using NMR and X-ray crystallography we to be resolved if backside ubiquitin binding is also important for show that the R6BD of Rad18 inhibits this activity by competing the activity of these enzymes. Only Ube2g2 has been shown to with ubiquitin for a noncovalent “backside” binding site on Rad6. function via an entirely different mechanism that results in long Our findings provide mechanistic insights into how E3 enzymes can ubiquitin chains being formed directly on its active site cysteine regulate the ubiquitin conjugation process. when in complex with the E3 enzyme gp78 (20–22). Although much of the specificity of the conjugation process is provided biochemistry ∣ translesion synthesis ∣ crystallography ∣ by E2 enzymes, E2/E3 enzyme complexes are required for full BIOCHEMISTRY NMR spectroscopy activity and specificity in vivo. Rad6 and its human homologues, Rad6a/b, are particularly biquitin conjugation to a lysine residue on a target is cata- important E2 enzymes that have been shown to be involved in Ulyzed by an E1, E2, E3 enzyme cascade. The E1 is required DNA damage tolerance (DDT), histone modification, and pro- for activation of the E2, but the E2/E3 complex is the active teasomal degradation, although other functions of Rad6 may ex- ligase that transfers the ubiquitin moiety to the target. Within this ist (23). DDT has been extensively characterized as a two-step ligase complex, over 30 E2 enzymes provide the enzyme activity pathway. In the first step, Rad6/Rad18 monoubiquitinate PCNA while hundreds of E3s define the target specificity (1). Ubiquitin on K164 at stalled replication forks to signal for recruitment of conjugation can take several different forms. The most simple is damage-tolerant polymerases. Next, Ubc13/MMS2/Rad5 can monoubiquitination, but because ubiquitin itself contains seven form K63-linked polyubiquitin chains on modified PCNA to in- lysine residues, it can be ubiquitinated to form many different itiate error-free DNA repair (6, 7). In this process Rad6 mono- linkages of ubiquitin chains. The different ubiquitin structures ubiquitinates only PCNA, even in vitro (24–26). Rad6a/b, with provide different molecular signals for processes such as protea- RNF20/40 (Bre1) as E3 ligase, will monoubiquitinate histone somal degradation, endocytosis, and DNA repair (2, 3). H2B in human cells on K120 (27) as an essential step prior to Mechanistic studies have revealed that ubiquitin chains can be H3 methylation by Set1 and Dot1. Finally, Rad6 together with assembled on a target in different ways. A single E2 enzyme can Ubr1 is associated with ubiquitin chain formation within the be responsible for synthesizing target-linked chains, as has been N-rule pathway. In this role, Rad6 is essential for the formation shown for CDC34 with the Skp1/Cul1/F-box protein E3 enzyme of K48 ubiquitin chains on bearing an N degron, which (4). Alternatively, two E2 enzymes can act sequentially on a single marks the target for proteasomal degradation (28). Interestingly, target to form specific chains, with one E2 enzyme initiating Rad18, Bre1, and Ubr1 all contain additional Rad6 recognition the chain formation and another extending the chains. Such a me- sites outside a canonical RING interface (27, 29, 30). The func- chanism has been shown for modifications of yeast Anaphase tional significance of Rad6 has been firmly established, but the promoting complex/cyclosome (APC) targets by Ubc4 then Ubc1 molecular mechanisms of Rad6’s activity are not well understood. (5) and modification of PCNA by Rad6 with E3 Rad18, then Here we analyze the structure and activity of Rad6b. We show Ubc13/MMS2 with E3 Rad5 (6, 7). To perform these modifica- that Rad6b is capable of forming ubiquitin chains via a non- tions, isolated E2 enzymes may be highly specific for monoubi- quitination or different linkages of ubiquitin chains. Ube2S, Author contributions: R.G.H., A.H., R.B., and T.K.S. designed research; R.G.H. performed E2-25K, and Ubc13/MMS2 are specialized enzymes that catalyse the enzyme assays and crystallography; A.H. performed the NMR experiments; R.G.H., the formation of K11, K48, and K63 chains, respectively (8–11), A.H., R.B., and T.K.S. analyzed data; and R.G.H., R.B., and T.K.S. wrote the paper. whereas UbcH5c can form many linkages of ubiquitin chains (12). The authors declare no conflict of interest . The molecular mechanisms of ubiquitin chain formation by This article is a PNAS Direct Submission. E2 enzymes are only starting to be understood. The specificity Freely available online through the PNAS open access option. of the E2 enzyme Ubc13, with the catalytically inactive E2 variant Data deposition: The crystallography, atomic coordinates, and structure factors have been (E2v) MMS2, for K63-linked ubiquitin chain synthesis has been deposited in the , www.pdb.org [PDB ID codes: Native Rad6b, 2yb6; explained in atomic detail (13). MMS2 simultaneously binds to Rad6b-Rad18 (R6BD) complex, 2ybf]. Ubc13 and orients an acceptor ubiquitin molecule such that only 1R.G.H. and A.H. contributed equally to this work. K63 is brought into close proximity to the ubiquitin thioester con- 2To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. jugate of Ubc13 (13). Interestingly chain formation by the human This article contains supporting information online at www.pnas.org/lookup/suppl/ E2 enzyme UbcH5 with E3 Brca1 depends upon a noncovalent doi:10.1073/pnas.1017516108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1017516108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 30, 2021 covalent interaction with ubiquitin. Rad18 inhibits this activity Some monoubiquitination of Rad6 was detected but not Rad6- during PCNA monoubiquitination because its major interaction linked ubiquitin chains (Fig. S1B), so we interpret that most of surface with Rad6 can compete with ubiquitin binding and pre- the chains are free in solution. We cannot exclude some level of vent ubiquitin chain formation by Rad6. Our results provide me- Rad6-linked ubiquitin chains. When Rad6b was analyzed in the chanisms by which different E3 enzymes can produce different absence of reducing agents, we observed thioester formation molecular signals with the same E2 enzyme: Rad6 is able to form (Fig. S1B), but no chains are formed on the active site cysteine target-linked ubiquitin chains, presumably catalyzed by appropri- of Rad6b. ate E3 enzymes, but is directed toward PCNA monoubiquitina- tion by Rad18. The Mechanism of the Chain Formation Is via a Noncovalent Interac- tion with Ubiquitin. To understand the mechanism of ubiquitin Results chain formation by Rad6 we used NMR to study the interactions Rad6b Can Form Ubiquitin Chains. When E2 enzymes can form of Rad6 with ubiquitin. Rad6 has previously been shown to ubiquitin chains in vivo, they typically retain some activity for ubi- bind noncovalently to ubiquitin as well as forming the covalent quitin chain formation in solution, even in the absence of an E3 thioester intermediate that is conserved among all catalytic E2 enzyme and a target. We studied if isolated Rad6b has the ability enzymes (31). We followed both events with NMR using the pre- to form ubiquitin chains in solution, using an in vitro reconsti- viously published assignment of Rad6b that was applicable to our tuted enzyme reaction. Rad6b showed a clear intrinsic ability to protein preparations (31, 32). The thioester formation on Rad6 form ubiquitin chains (Fig. 1A and Fig. S1A). The specificity of was followed by incubating 15N-labeled Rad6b with ubiquitin, the ubiquitin chain linkages was studied using of ubiqui- ATP∕Mg2þ, and E1 activating enzyme. Within 1 h after addition tin with a single lysine mutated to arginine. The activity was of ATP,changes in chemical shifts were observed for residues sur- modestly reduced in all of the mutants compared with wild-type rounding the active site cysteine of Rad6b, consistent with Rad6b ubiquitin, but the K11R showed a more dramatic effect: forming a thioester bond with ubiquitin (Fig. 1C and Fig. S2A). The molecular weight of the ubiquitinated species was strongly We performed a structural characterization of the noncovalent reduced and a diubiquitin species was no longer visible. This is complex as a basis for studying its role in ubiquitin chain forma- indicative of Rad6b forming mixed ubiquitin chains, with a fairly tion. In the absence of a high-resolution structure, we validated strong preference for K11-linked chain formation (Fig. 1B). published NMR titrations (31), then performed a data-driven

Fig. 1. Rad6b can form ubiquitin chains via a noncovalent interaction with ubiquitin. (A) Time-course experiment of ubiquitin chain formation by Rad6b. Antiubiquitin Western blot using an in vitro system containing purified E1 (90 nM), His-Rad6b (3 μM), ubiquitin (12 μM), and ATP∕Mg2þ (3 mM∕ 10 mM). The time points are as follows: 8 h, 0 h, 30′, 1 h, 2 h, 4 h, and 8 h (lanes 1–7). (B) Specificity of Rad6-catalyzed chain formation. Antiubiquitin Western blot of in vitro ubiquitin chain-formation assays using E1 (90 nM), His-Rad6b (3 μM), and wild-type ubiquitin or single lysine to arginine point mutants of ubiquitin (12 μM) after 8 h. The ubiquitin mutants are as follows: WT, WT, K6R, K11R, K27R, K29R, K33R, K48R, and K63R (lanes 1–9). The experiment re- veals a preference of K11-linked ubiquitin chains. (C) Rad6b forms a thioester bond with ubiquitin in the presence of E1 and Mg2þ∕ATP. NMR experiments showed significant CSPs of residues D50, G51, T69, N80, I87, C88, L89, D90, I91, L92, Q93, A122, and N123 (colored green) near the active site cysteine, C88, following thioester formation of Rad6b with ubiquitin. (D) The Rad6b- ubiquitin noncovalent complex. Cartoon representation of the lowest energy docking solution of Rad6b-ubiquitin noncovalent complex. Significant CSP of Rad6b (>0.05; residues 22–26, 38–39, 41, 51–52, 142, and 146–147) and ubiqui- tin (CSP > 0.025; residues 7–8, 13–14, 32, 41–42, 47–49, and 68–72) are high- lighted in green and blue. Residues G23 and T52 are highlighted on the cartoon representation in red. The affinity of the interaction is estimated to be approximately 600 μM. (E) A structural superposition of UbcH5/ubiquitin [cyan; PDB ID code 2FUH(14)], MMS2/ubiquitin [green; PDB ID code 2GMI (13)], and Rad6b/ubiquitin (blue). The mode of ubiquitin binding is highly similar between these E2 and E2v enzymes. (F) Point mutations at “backside” of Rad6b interfere with ubiquitin chain formation. The point mutants were selected to interfere with ubiquitin binding but not Rad18 binding, based on our structural data (see below). Assay follows chain formation after 4 h with purified E1 (90 nM) and ubiquitin (12 μM) and a 2-fold dilution series of wild- type His-Rad6b or a G23A T52R His-Rad6b mutant, analyzed by antiubiquitin Western blotting. The Rad6b concentrations are as follows: 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0 μM (lanes 1–10). (G) The back side G23A T52R Rad6 mutant is not defective in E1 interaction. Thioester formation experiment assay with His-Rad6b (3 μM), ubiquitin (12 μM), and a 3-fold dilution series of E1, analyzed by anti-His (Rad6) Western blot after 10 min under nonreducing conditions. The E1 concentrations were as follows: 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0 nM, 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.4 nM, and 0 nM (lanes 1–14). No significant isopeptide bond formation was observed under these conditions. (H) Model showing the chain-formation activity of Rad6b depends upon a covalent and noncovalent interaction with ubiquitin.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017516108 Hibbert et al. Downloaded by guest on September 30, 2021 docking approach to generate models of the complex (33). The plexes only monoubiquitinate PCNA, we studied whether Rad18 significant chemical shift perturbations (CSPs) in the NMR titra- inhibits the chain-formation activity of Rad6b upon forming a tions mapped to β-strands β1-3 and helix α4 on Rad6b and to binary Rad6/Rad18 complex. We tested the chain-forming activ- β-strands β1-5 of ubiquitin (Fig. 1D and Fig. S2B). Our highest ity of Rad6 in the presence of Rad18. Remarkably, the chain scoring docking models pack the hydrophobic core of ubiquitin, formation is abrogated when Rad18 is present in stoichiometric surrounding Ile44, against a small hydrophobic patch on the amounts (Fig. 2B). “backside” of Rad6b, around Val39 and Phe41 (Fig. 1D). Inter- estingly, when our model is compared with existing structures The Rad18 (R6BD) Competes with Ubiquitin Binding and Ubiquitin of E2s and E2 variants making noncovalent complexes with ubi- Chain Formation. To understand the mechanism of this inhibition quitin (13, 14), we find the complexes are highly similar to each of chain formation by Rad18, we studied the interactions between other and differ only by small rotations in the orientation of the Rad6 and Rad18. Rad18 recognizes Rad6 via an N-terminal ubiquitin (Fig. 1E). This confirms that the backside of Rad6b RING domain and a separate C-terminal Rad6 binding domain interacts with ubiquitin via a canonical mode of interaction. (R6BD) (Fig. 3A) (29, 34). Because the RING-E2 interaction is To understand the functional relevance of the Rad6b-ubiquitin highly conserved among E2-E3 complexes regardless of their complex in ubiquitin chain formation, we constructed single point chain-formation activity, and Rad18 binds via this canonical mutants on the backside of Rad6b to perturb the complex, based interaction, we studied the interaction of Rad6 with the Rad18 on our structural data, and tested the recombinantly expressed (R6BD) using NMR titrations. The observed CSPs mapped proteins in our in vitro chain-formation assay. Both a G23R the binding site of the R6BD at the backside of Rad6b, opposite and T52A mutation (Fig. 1D) show reduced activity for ubiquitin to the active site cysteine, C88 (Fig. 3 B and C and Fig. S2C), chain formation (Fig. S1C) and a G23R T52A double mutant of with major shifts on β-strands β1, β2, and β3 and the C-terminal Rad6b was further compromised in ubiquitin chain formation residues of Rad6b. The titrations are consistent with a 62-μM (Fig. 1F), without any detectable effect on E1 interaction affinity and fast-on/fast-off rates for the interaction that were (Fig. 1G). We conclude that the noncovalent backside interaction observed using SPR (Fig. S3A). is required for chain formation (Fig. 1H). We solved the crystal structure of Rad6 alone and in complex with the R6BD peptide as a basis to understand the interaction Rad18 Inhibits the Rad6′s Chain-Formation Activity. We studied the in atomic detail (Fig. 3 C and D, Table 1, and Fig. S4). In both activity of Rad6 toward PCNA. Consistent with previous reports structures the Rad6b has the canonical E2 fold, and the two

(24–26), Rad6 will modify PCNA only when in complex with monomers are highly similar (Fig. S4). The R6BD structure is BIOCHEMISTRY E3 ligase Rad18, with no evidence for ubiquitin chains formed that of a kinked helix with residues Ser348-Arg358 forming a by the Rad6/Rad18 complex (Fig. 2 A and C). Because Rad6b long α-helix and residues Lys341-Lys344 forming a second helix is capable of forming ubiquitin chains, but Rad6/Rad18 com- rotated by about 60° with respect to the first. Consistent with the NMR data, the peptide interacts with the backside of Rad6b, via a series of hydrogen bonds, salt bridges, and van der Waals contacts (Fig. 3 C and D). The isolated R6BD peptide forms loosely folded helices in solution (Fig. S3 B and C and Table S1). The R6BD adopts the more compact structure upon complex formation. Our structural studies show that the major binding site of Rad18 (R6BD) on Rad6b overlays with the noncovalent ubiquitin interaction site that is important for ubiquitin chain formation (Fig. 4A). Therefore we postulated that the R6BD of Rad18 could compete with the ubiquitin chain-formation activity of Rad6 by preventing binding to ubiquitin. Our affinity estimates for the individual interactions imply that the Rad18 (R6BD) should efficiently compete with ubiquitin for binding to the back- side of Rad6b. We tested the competition between the two inter- actions using our NMR assay. This experiment showed that the R6BD can compete with ubiquitin for Rad6 binding (Fig. 4B). Next, we studied if R6BD has an effect on the Rad6’s chain- formation activity. We followed the ubiquitin chains formed by Rad6b alone or in the presence of R6BD. The R6BD peptide could compete for Rad6’s ubiquitin chain-formation activity (Fig. 4 C and D). As a control, we used an R6BD* peptide with Fig. 2. Rad18 inhibits Rad6’s ubiquitin chain-formation activity to direct four point mutations (H346A, L353A, V354A, and A357R) that A Rad6 toward PCNA monoubiquitination. ( ) Time-course experiment of maintained the same overall charge as the native peptide and PCNA monoubiquitination by Rad6b or Rad6b/Rad18 complexes. Anti-PCNA contained the same number of lysine residues, but bound with a Western blot following an in vitro assay containing purified E1 (90 nM), Rad6b (20 μM), or Rad6b/Rad18 (20 μM), ubiquitin (12 μM) and PCNA (3 μM). lower affinity. R6BD* showed a highly reduced inhibition of the The following time points were used: 0, 1 h, 4 h, 16 h, 0, 1 h, 4 h, and 16 h ubiquitin chain-formation activity (Fig. 4 C and D and Fig. S1D). (lanes 1–8). The Rad6/Rad18 dependent PCNA monoubiquitination is almost A small inhibition remained, however, probably because R6BD* complete under these conditions, with no evidence for chain formation on still has some affinity for Rad6b, or because peptide modification PCNA. Rad6b shows little detectable activity for PCNA in the absence of itself competes for ubiquitin in the assay. As an additional control Rad18. (B) The full-length Rad18 inhibits Rad6-mediated ubiquitin chain we studied if the secondary interaction site of chain-forming E3 formation. Time-course experiment of ubiquitin chain formation by E1 μ μ ligase Ubr1, the basic residues-rich (BRR) domain, was capable (90 nM), ubiquitin (12 M), and His-Rad6b (3 M) alone, or reconstituted of competing with Rad6b for ubiquitin binding and ubiquitin with stoichiometric His-SUMO-Rad18, followed by antiubiquitin Western blotting. The following time points were used: 3 h, 0, 20′,1h,3h,0,20′,1h, chain formation. We show that the BRR binds to the same site and 3 h (lanes 1–9). Rad18 was expressed in the absence of Rad6b and recon- as ubiquitin on the backside of Rad6b, but with only a millimolar stituted into the reaction. Significant ubiquitination of Rad18 was detected. affinity. The BRR is not able to compete for ubiquitin binding (C) Cartoon showing PCNA monoubiquitination by Rad6/Rad18 complexes. or ubiquitin chain formation (Fig. S5). In summary, the Rad18

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Fig. 3. The interaction of R6BD with Rad6b. (A) Domain structure of Rad18 with the amino acid sequence of the R6BD indicated. (B) NMR spectrum (1H-15N- HSQC) of Rad6b alone (black) with increasing amounts of R6BD to a final Rad6b:R6BD molar ratio of 1∶4 (red). (C) Crystal structure of the complex of R6BD and Rad6b. R6BD (orange) is a kinked helix that binds to the back of Rad6b. Significant (>0.1) CSPs of Rad6b (residues 23–27, 37–41, 44, 50–52, 54–57, 146, and 148–149) are highlighted in green. The crystal structure is highly consistent with the NMR titration data. (D) Details of the Rad6b interaction with R6BD. The interaction of the peptide with Rad6b is stabilized by potential hydrogen bonds to Tyr342, Arg343, and His346 at the kinked N terminus of the peptide and Tyr361 at the C terminus, although this residue may be somewhat mobile because it is less well resolved in the electron density. The interaction is also stabilized by a number of hydrophobic interactions including Val354 and Ala357, which are buried in a hydrophobic patch on Rad6b surrounding Val39 and Phe41. Residues at the interface on R6BD and Rad6b are colored yellow and green, respectively.

(R6BD) can compete with the activities of Rad6 for ubiquitin (1). Consistent with this, Rad18 RING domain mutants show re- binding and ubiquitin chain formation under these conditions, duced activity in vivo (35, 36). The R6BD interaction is required but the Ubr1 (BRR) cannot. in vivo in higher eukaryotes (36), and in our in vitro reactions. We studied the effect of mutating the R6BD on the activity of The interaction is also important in yeast because cells that full-length Rad18. We produced full-length Rad18* protein that produced Rad18 with a truncated R6BD showed increased sen- contained the same four-point mutants in the R6BD domain sitivity to UV irradiation (29). Our high-resolution structure of (H346A, L353A, V354A, and A357R) and compared the activity the Rad6-R6BD complex is highly consistent with the mapping of this protein with wild-type Rad18 in the Rad6-dependent experiments in the yeast study but reveals that their in vivo modification of PCNA. Mutations in the R6BD strongly reduce constructs (29) still contained a critical part of the R6BD. It is the PCNA ubiquitination because an intact R6BD is required likely that constructs lacking the complete R6BD will be even for transfer of ubiquitin onto PCNA (Fig. S1E). Consequently more sensitive to UV light. we do not observe ubiquitin chains on PCNA (Fig. S1E). Finally, We demonstrate that human Rad6b can form ubiquitin chains, we studied the ability of Rad18* to inhibit the chain-formation as has been shown for Rad6 (37). We activity of Rad6. In contrast to the wild-type protein, our mutant show that chain formation by human Rad6 depends upon a Rad18 is no longer able to inhibit the chain-formation activity noncovalent interaction with ubiquitin, using mutagenesis as of Rad6 (Fig. S1F), confirming that a functional Rad18 (R6BD) well as R6BD peptides that inhibit the ubiquitin interaction and is required to inhibit Rad6’s chain-formation activity. the chain-formation activity of Rad6 in a dose-dependent man- ner. This places the chain-formation activity of Rad6 in the same Discussion class as several other ubiquitin E2s, SUMO E2s, and E2/E2v com- We show that Rad6 has an intrinsic capability for ubiquitin chain plexes (13–15, 17). Models that use such a noncovalent interac- synthesis that depends upon a noncovalent interaction with tion are attractive because they provide an explanation for how ubiquitin. Rad18 is capable of modulating this activity by compet- the E2 is associated with the end of the growing ubiquitin chain, ing with the noncovalent ubiquitin interaction. We propose that which becomes increasingly distant from the original conjugation Rad18 utilized this mechanism to direct Rad6 toward monoubi- site on the target as the length of the chain increases. quitination rather than chain formation on PCNA. In contrast to Rad6, Ube2g2 has been shown to synthesize Rad18 can recognize Rad6 via its RING and R6BD domains. ubiquitin chains via a very different mechanism from other E2s, RING-E2 interactions are highly conserved among RING E3 because it forms long ubiquitin chains on its active site cysteine, and a functional interaction is a prerequisite for activity when in complex with E3 enzyme gp78 (20). Ube2g2 utilizes a

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017516108 Hibbert et al. Downloaded by guest on September 30, 2021 Table 1. Data collection and refinement statistics AC Rad6b* Rad6b + Rad18 (339–366)* Data collection Space group P43212 P6522 Cell dimensions a, b, c (Å) 48.1, 48.1, 124.8 58.2, 58.2, 167.1 α, β, γ (°) 90.0, 90.0, 90.0 90.0, 90.0, 120.0 Resolution (Å) 44.84–1.50 24.92–2.00 (1.58–1.50) (2.11–2.00) R merge 0.086 (0.431) 0.128 (0.718) Mean [ðIÞ∕σðIÞ] 13.0 (4.0) 16.0 (4.7) Completeness (%) 98.1 (97.2) 99.3 (95.5) Redundancy 5.9 (6.0) 12.9 (12.1) Refinement Resolution (Å) 1.50 2.00 B No. reflections 23,655 11,963 D R ∕R 0 166∕0 205 0 201∕0 242 work free . . . . No. atoms Protein 1,198 1,390 Ligand/ion 5 15 Water 135 85 B factors Protein 21.6 29.8 Ligand/ion 29.0 42.6 Water 33.3 16.8 rms deviations Bond lengths (Å) 0.0118 0.008 Bond angles (°) 1.409 0.967 *One crystal. BIOCHEMISTRY

similar mode of interaction with gp78, to the complex between Fig. 4. Ubiquitin chain formation by Rad6b is inhibited by competition Rad6 and Rad18 (21, 22). The gp78 peptide forms a continuous with Rad18 (R6BD). (A) The binding sites of ubiquitin and R6BD on Rad6b helix on the backside of Ube2g2 and the binding location of this overlap. The crystal structures of ubiquitin with UbcH5 (cyan) and MMS2 helix is similar to the R6BD, but it is oriented approximately (green) and our docking-based model of the Rad6b-ubiquitin complex (blue) perpendicular to that of Rad18 on Rad6. Ube2g2 has evolved were superimposed upon the crystal structure of Rad6b in complex with with differences close to its active site, and allosteric activation Rad18 (R6BD) using only the E2s for the superposition. The cartoon represen- tations of ubiquitin are shown as a semitransparent representation. Rad6b of these residues upon gp78 interaction activates its unique me- (blue) and Rad18 (R6BD) are shown as opaque cartoons. (B) R6BD can com- chanism (20, 21). pete for backside ubiquitin binding. 1H-15N-HSQC peaks of 15N-labeled We show that Rad18 can compete for Rad6’s activity toward Rad6b were studied that had been perturbed in a different direction ubiquitin chain formation by competing with the noncovalent upon addition of ubiquitin (ii) and R6BD (i) during the separate titrations. interaction of Rad6 with ubiquitin. In vivo Rad18 is itself mod- A 15N-labeled sample of Rad6b (200 μM) was preincubated with a 3-fold ified with K48-linked ubiquitin chains that are recognized by the molar excess of ubiquitin (ii) and R6BD was titrated into the sample to the proteasome (34). This modification is likely to be indirect, with same final concentration as ubiquitin (iii). These peaks were observed to iii the chains formed by other E2/E3 enzymes, because a functional move to the same final positions ( ) as the single titration of R6BD into Rad6b (i), confirming that R6BD can compete with ubiquitin for binding to Rad18-RING domain is not required. Rad6b. (C) Time-course experiment showing that R6BD can inhibit Rad6- Rad6 is an abundant E2 enzyme, present in micromolar con- mediated ubiquitin chain formation. The assay contains purified E1 (90 nM), centrations in vivo (38) comparable to our assays. The chain- His-Rad6b (20 μM), and ubiquitin (20 μM) alone or together with R6BD formation activity of Rad6 that we detect is modest; however, (100 μM) or a mutated R6BD* (128 μM; H346A, L353A, V354A, and A357R) one or more specific E3 enzymes would be expected to activate peptide, followed by antiubiquitin Western blotting. The following time Rad6’s ubiquitin chain-formation activity toward specific targets. points were used: 0, 30′,2h,8h,0,30′,2h,8h,0,30′, 2 h, and 8 h (lanes – D Among the E3 partners of Rad6b, a role of Rad6-mediated 1 12). Both peptides undergo significant monoubiquitination. ( ) A concen- tration series of R6BD shows dose-dependent inhibition of Rad6-mediated ubiquitin chain formation in the Rad18 and Bre1 pathways is ubiquitin chain formation. The assay contains purified E1 (90 nM), His-Rad6b unlikely because Rad6 is currently believed to exclusively mono- (3 μM), and ubiquitin (12 μM) alone or together with a twofold dilution ubiquitinate the targets in these systems (7, 27). Interestingly, series of R6BD (lanes 3–7: 64 μM, 128 μM, 256 μM, 512 μM, and 1,024 μM) Rad6/Ubr1-dependent ubiquitin chains are directly produced by or R6BD* (lanes 9–13: 64 μM, 128 μM, 256 μM, 512 μM, and 1,024 μM) at the N-rule degradation pathway (39), so it is credible that Rad6’s 8 h. R6BD shows a far greater activity than R6BD* for inhibiting the noncovalent interaction with ubiquitin is used to synthesize the chain-formation activity of Rad6. chains. We studied whether the BRR secondary interaction site of Ubr1 was capable of modulating Rad6b-mediated ubiquitin It is likely that more functions of Rad6 remain to be identified, chain formation. This domain bound to the backside of Rad6b, on previously undescribed targets or with different E3 ligases, but the affinity was so low that it was not able to compete with ubiquitin binding or ubiquitin chain formation, in contrast to the which could utilize the chain-formation propensity of Rad6. R6BD of Rad18. It is plausible that the Ubr1 (BRR)-Rad6b in- Because Rad18, Bre1, and Ubr1 all bind to Rad6 via unusual bi- teraction is dynamic during the activity of Ubr1 in N-rule degra- valent interactions, E3 enzymes that bind via weaker interactions dation and that ubiquitin chains are synthesized via the backside that require only a RING domain may not have been discovered interaction of Rad6, although we cannot rule out a higher Rad6b- yet. Consistent with this, a recent reconstruction of the ubiquitin BRR affinity in the context of the full-length proteins. It is also network in yeast identified Rad6 as a major hub, with many possible that Rad6/Ubr1 complexes form ubiquitin chains on Golgi-, vesicle-, or endosome-associated roles for Rad6 that N-rule targets via a yet unknown mechanism. are not explained by currently known targets of Rad6 (23). These

Hibbert et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 30, 2021 roles may use Rad6b to mark a target for proteasomal degra- formed on a Bruker Avance2 750-MHz NMR and followed by 1H-15N hetero- dation, because we observed some specificity for K11-linked nuclear sequential quantum correlation (HSQC) spectra. Docking of the ubiquitin chains. noncovalent Rad6b/Ub complex was performed using the Haddock Web We find that the chain-formation activity of Rad6 can be server (33). Crystals of Rad6 and the Rad6 (R6BD) complex were grown by inhibited by E3 complex formation with Rad18 and predict vapor diffusion and solved using molecular replacement. The final model R ∕R 16 6%∕20 5% that other E3 enzymes can promote or inhibit chain formation was refined to work free of . . at 1.5-Å resolution (Rad6) or R ∕R 19 9%∕24 2% in different biological contexts. This functional diversity of a work free of . . at 2.0-Å resolution (Rad6-R6BD complex). single E2 is not surprising given the low number of E2 enzymes Further details for all experiments are available in SI Methods. compared with E3s and targets. Understanding how E3s modu- late the activity of E2s toward chain formation on a particular ACKNOWLEDGMENTS. We are grateful to European Synchrotron Radiation target becomes an exciting area for future research. Facility beam line scientists, Henk Hilkman for peptide synthesis, Dene Littler and Alex Faesen for crystallography data collection, and Anastassis Methods Perrakis and Robbie Joosten for advice during data processing and refine- Rad18-R6BD (339-366), R6BD* (as R6BD with H346A, L353A, V354A, and ment. Valerie Notenboom provided useful discussions and Anastassis Perra- A357R), and Ubr1-BRR (1013-1028) were produced as synthetic peptides. kis, Judith Smit, and Peter Krijger provided comments on the manuscript. Rad6b, Rad18, Uba1, Ubiquitin, and PCNA were overexpressed in Escherichia Funding was from the European Union SPINE2COMPLEXES (T.K.S. and coli and purified by successive chromatography steps. In vitro ubiquitination R.B.), the Dutch Organization for Scientific Research (NWO) TOP grant assays were performed using conditions stated in the figure legends, resolved (R.B.), and European Union Role of Ubiquitin and Ubiquitin-like Modifiers on SDS-PAGE gels, immunoblotted, and analyzed. NMR titrations were per- in Cellular Regulation (T.K.S.).

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