876 volume 19 number 9 SePTember 2012 nature structural & molecular biology

1The Beatson Institute for Cancer Research, Glasgow, UK. 2These authors contributed equally to this work. Correspondence should be addressed to D.T.H. (d.huang@beatson.gla.ac.uk).

Received 21 May; accepted 8 August; published online 14 August 2012; doi:10.1038/nsmb.2379

state intermediate15, and an aspartate side chain may suppress the pKa of lysine on a substrate16. These findings have led to the proposal that RING E3 binding may organize the E2’s active site through allosteric effects to enhance substrate ubiquitination8,9.

Several RING E3s must homo- or heterodimerize to form active ligases. For example, RAD18, RNF4, TRAF6, IDOL and the baculoviral inhibitor of apoptosis repeat-containing protein (BIRC) family of RING E3s form homodimers, whereas others, like MDM2, BRCA1 and RNF2, heterodi-merize with inactive RING domains, namely MDMX, BARD1 and BMI1, respectively (reviewed in ref. 13). Although the structures of dimeric RINGs differ slightly, dimerization is achieved by RING–RING interac-tions and by residues flanking the N- and C-terminal regions of the RING domain. Mutations that disrupt RING dimerization reduce ubiquitination activity17–20. Recent studies on BIRC-family RING E3s and RNF4 showed that RING dimerization is required for E2~Ub binding18,21. For RNF4, one subunit of the RING domain dimer is proposed to bind E2 and the C-terminal tail of the second subunit to interact with the Ile44 surface of the corresponding donor Ub to facilitate Ub transfer18. The importance of the C-terminal tail of the RING dimer is supported by a previous study on MDM2–MDMX, where inactive MDM2 C-terminal tail mutants were rescued by heterodimerization with wild-type MDMX22. Several models describing how RING E3s promote Ub transfer have been proposed on the basis of a wealth of biochemical data, but there is currently no available structure of a RING E3–E2~Ub complex.

BIRC proteins play a major part as negative regulators of cell death, and several members of this protein family, including BIRC2 and BIRC3, contain a C-terminal RING domain that homodimerizes to form an active E3 (refs. 17,21). The E3 activity is required for ubiquitination and subsequent degradation of critical apoptotic-pathway components,

Post-translational modification by Ub is an important regulatory mech-anism for a wide variety of cellular processes1,2. Ub is activated and conjugated to substrate by the consecutive actions of a ubiquitin-activating enzyme (E1), an E2 and an E3 (ref. 1). E1 activates and forms a covalent thioester intermediate with the C terminus of Ub in an ATP-dependent manner. Ub is then transferred to E2, resulting in a covalent thioester linkage between E2’s catalytic cysteine and Ub’s C terminus (E2~Ub). E3s promote transfer of Ub from E2 to the amino group of a substrate lysine and generally contain E2 and substrate-binding domains to facilitate this process. Several classes of E3s have been defined based on the mechanism of transfer: HECT, RING, U-box and RING-in-between-RING3,4. For RING E3s, the largest class5, Ub is transferred directly from E2~Ub to substrate without the formation of a covalent E3~Ub intermediate.

How RING E3s promote Ub transfer is unclear. In the absence of an E3, E2~Ub is able to catalyze the transfer of donor Ub (conjugated Ub) to an acceptor Ub, free amine or substrate4,6–11, but addition of a RING E3 massively enhances the rate4,8,9,11. In several structures of RING E3–E2 complexes, the RING domain binds E2 at a region distal to the E2’s active site, precluding direct modification of the active site by the RING E3 (refs. 12,13). Only biochemical analyses provide some insight into how RING E3s promote Ub transfer. Recent studies have shown that interactions between the Ile44 surface of the donor Ub and E2 a3’s residues are important for donor Ub transfer in the absence and presence of RING E3s9,10. Notably, mutations on the surface of the E2 UbcH5B, which are distal from both the RING domain–binding site and active site, influence the ability of RING E3s to stimulate Ub trans-fer8,14. In addition, mutational analyses of several E2s have identified highly conserved key residues near E2’s active site that are critical for catalysis: an asparagine side chain may stabilize the oxyanion transition

BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimerHao Dou1,2, Lori Buetow1,2, Gary J Sibbet1, Kenneth Cameron1 & Danny T Huang1

certain ring ubiquitin ligases (e3s) dimerize to facilitate ubiquitin (ub) transfer from ubiquitin-conjugating enzyme (e2) to substrate, but structural evidence on how this process promotes ub transfer is lacking. Here we report the structure of the human dimeric ring domain from birc7 in complex with the e2 ubcH5b covalently linked to ub (ubcH5b~ub). the structure reveals extensive noncovalent donor ub interactions with ubcH5b and both subunits of the ring domain dimer that stabilize the globular body and c-terminal tail of ub. mutations that disrupt these noncovalent interactions or ring dimerization reduce ubcH5b~ub binding affinity and ubiquitination activity. moreover, nmr analyses demonstrate that birc7 binding to ubcH5b~ub induces peak-shift perturbations in the donor ub consistent with the crystallographically-observed ub interactions. our results provide structural insights into how dimeric ring e3s recruit e2~ub and optimize the donor ub configuration for transfer.

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Donor ub–ubcH5b interactionsOur structure shows detailed molecular interactions between a donor Ub covalently linked to an E2 in the presence of a RING E3. Unlike other published E2~Ub structures, our E2~Ub conjugate appears poised for transfer. The Ile44 patch of Ub makes extensive noncovalent interac-tions with the entire length of UbcH5B’s a3 (Fig. 2a). Recently, E2’s a3–Ub Ile44 surface interactions have been identified to be important for Ub transfer by the E2s Ube2S and Cdc34 (refs. 9,10); for Cdc34, an S129L mutation on a3 of the E2, combined with an I44A muta-tion on Ub, rescues Ub transfer defects associated with the individual mutants. In our structure, Ser108 of UbcH5B (corresponding to Ser129 in Cdc34) interacts with Ile44 of Ub. To assess the importance of the observed interactions, we performed in vitro single-turnover lysine discharge4, autoubiquitination and SMAC ubiquitination assays under pulse-chase conditions to eliminate mutational effects from E1-catalyzed UbcH5B~Ub formation (Supplementary Note). Mutations of key resi-dues on UbcH5B’s a3 (K101E, L104A, S108R and D112A) and Ub’s Ile44 surface (L8D, R42E, I44A, G47E, Q49E and H68A V70A) cause defects in Ub transfer (Fig. 2b,c and Supplementary Fig. 2a–g). In addition, Ub’s L8D and I44A and UbcH5B’s S108R greatly reduce binding affinity when incorporated into UbcH5BS~Ub (Table 1).

The observed UbcH5B~Ub conformation precludes binding of the donor Ub to UbcH5B’s backside in the presence of BIRC7, because both interactions use the same Ub surface. To investigate the role of UbcH5B’s backside binding, we tested UbcH5B S22R in BIRC7-catalyzed assays. UbcH5B S22R displayed similar activity as wild-type in lysine discharge assays but was strongly defective in polyubiqui-tinating BIRC7 and SMAC (Fig. 2b and Supplementary Fig. 2d,e),

including caspases and second mitochondrial activator of caspases (SMAC), for activation of NF-kB signaling and for degradation of other proteins (reviewed in ref. 23). To gain insights into the role of RING dimerization and to understand how RING E3s interact with E2~Ub to promote Ub transfer, we used the BIRC RING E3s as a model system and determined the crystal structure of the dimeric BIRC7 (also known as melanoma inhibitor of apoptosis protein and Livin) RING domain in complex with UbcH5B covalently linked to Ub’s C terminus. Along with NMR and biochemical analysis, the data reveal that combined interac-tions between UbcH5B, donor Ub and the BIRC7 RING dimer are cru-cial for Ub transfer, elucidate the roles of catalytically important residues outside E2’s active and RING-binding sites, and identify noncanonical interactions between the Ile36 surface of Ub and the BIRC7 RING dimer. We propose that, as suggested for several RING E3s8,9,18, BIRC7 pro-motes activity by stabilizing an optimal E2~donor Ub arrangement for Ub transfer. Here, to our knowledge, we show the first detailed structural evidence supporting this hypothesis.

resultscharacterization of the birc7 ring–ubcH5b~ub complexSeveral homodimeric RING E3s, such as RNF4 and BIRC3, have dis-tinct binding preferences for E2~Ub as compared to E2 (refs. 18,21). To determine whether BIRC7 has similar preferences, we measured binding affinities of the BIRC7 and BIRC3 RING domains for UbcH5B~Ub and UbcH5B by using surface plasmon resonance (SPR) analysis. Because E2 thioesterified with Ub rapidly dissociates into E2 and Ub in the presence of RING E3, UbcH5B’s catalytic Cys85 was mutated to serine to gener-ate a stable covalent ester linkage (UbcH5BS~Ub). Like BIRC3, BIRC7 showed a higher binding affinity for UbcH5BS~Ub than for UbcH5B (Table 1 and Supplementary Fig. 1), suggesting that the donor Ub con-tributes to the RING E3–E2~Ub interaction.

To investigate the donor Ub role in binding, we determined the structure of BIRC7 RING domain (BIRC7239–C) bound to UbcH5BS S22R N77A~Ub (UbcH5BRAS~Ub) to 2.18 Å (Fig. 1 and Table 2). We attempted to crystallize the BIRC7239–C bound to UbcH5BS~Ub but found that it dissociated to UbcH5B and Ub after 1–3 days; hence, we mutated Asn77 within the highly conserved E2 HPN motif15 to alanine to increase UbcH5BS~Ub stability. UbcH5B’s Ser22 was mutated to argi-nine to prevent backside binding with Ub14. The complex has a compact overall structure with two copies of the BIRC7239–C–UbcH5BRAS~Ub heterotrimer in the asymmetric unit (r.m.s. deviation of 0.43 Å for Ca atoms). For simplicity, we differentiate molecules from the two hetero-trimers with A or B subscripts (for example, BIRC7A or UbB; Fig. 1). The structures of UbcH5BRAS and Ub resemble previously published UbcH5B and Ub structures24 (r.m.s. deviation of 0.43 and 0.42 Å, respectively, for Ca atoms). The structure of BIRC7239–C is similar to the BIRC3 RING domain17 (r.m.s. deviation of 1.13 Å for Ca atoms), as an a-helix precedes the RING domain, and the C-terminal tails of BIRC7 from both subunits participate in RING dimerization (Fig. 1).

The canonical RING–E2 interactions described previously12,17 are conserved in our BIRC7239–C–UbcH5BRAS~Ub complex. Ub nestles along UbcH5B’s a3 and the BIRC7 RING domain within each subunit of the asymmetric unit. Notably, the C-terminal tail of BIRC7B packs against UbA, and the C-terminal tail of BIRC7A packs against UbB (Fig. 1). Ub’s Ile44 and Ile36 patches make extensive contacts with UbcH5B and the BIRC7 dimer, respectively. The C-terminal tail of Ub lies along the interface between UbcH5B’s a2 and a3, making numerous contacts with UbcH5B, the globular Ub body and the RING domain (Fig. 1). The quality of the electron density was better for subunit A of the BIRC7239–C–UbcH5BRAS~Ub complex, so we focus on this subunit and the C-terminal tail of BIRC7B.

Table 1 Dissociation constants (Kd) for interactions between RING E3 variants, UbcH5B and UbcH5BS~Ub variantsGST-immobilized protein Analyte Kd (mM)a

BIRC3541–C UbcH5B 300 ± 7

BIRC3541–C UbcH5BS~Ub 5 ± 4

BIRC7239–C UbcH5B N.M.

BIRC7239–C UbcH5BS~Ub 136 ± 2

BIRC7FL UbcH5B N.M.

BIRC7FL UbcH5BS~Ub 181 ± 29

BIRC7FL UbcH5BS I88A~Ub N.M.

BIRC7FL UbcH5BS S108R~Ub N.M.

BIRC7FL UbcH5BS D117A~Ub 191 ± 18

BIRC7FL UbcH5BS~Ub L8D N.M.

BIRC7FL UbcH5BS~Ub G35E N.M.

BIRC7FL UbcH5BS~Ub I36A N.M.

BIRC7FL UbcH5BS~Ub I44A N.M.

BIRC7FL V254A UbcH5BS~Ub N.M.

BIRC7FL V263R UbcH5BS~Ub N.M.

BIRC7FL I284A UbcH5BS~Ub N.M.

BIRC7FL R286A UbcH5BS~Ub N.M.

BIRC7FL F296H UbcH5BS~Ub N.M.

BIRC7FL D296–298 UbcH5BS~Ub N.M.

MDM2428–C–MDMX428–C UbcH5B N.M.

MDM2428–C–MDMX428–C UbcH5BS~Ub 130 ± 31

RNF21–114–BMI11–109 UbcH5B 91 ± 2

RNF21–114–BMI11–109 UbcH5BS~Ub 90 ± 2s.e.m. values are indicated. Number of replicates, representative sensorgrams and binding curves are shown in Supplementary Figure 1. Subscripts denote the range of residues incorporated in the variant. FL indicates full-length protein. N.M. indicates Kd was not measurable.aBecause of the weak binding, we could not saturate UbcH5B or UbcH5BS~Ub variant con-centrations for some of these measurements. The Kd values were estimated by fitting data obtained from steady-state affinity analyses of 0–180 µM UbcH5B or UbcH5BS~Ub variants.

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suppression during catalysis16. Mutation of Asp117 produces defects in Ub transfer mediated by BIRC7 (Fig. 2b and Supplementary Fig. 2d,e) and other RING E3s4,8,24. Notably, UbcH5BS D117A~Ub and UbcH5BS~Ub display similar binding affinities for BIRC7 (Table 1), which suggests that Asp117 may not be involved in E2~Ub recruitment but may instead have an alternative role in catalysis. Whether the observed E2 active site con-formation is optimized for transfer or is a product of the N77A mutation requires further investigation.

consistent with previous reports showing that backside binding is required for polyubiquitination14,24.

Residues 71–74 of the C-terminal tail of UbA are maintained by con-tacts with UbcH5BA’s a2, UbA’s Leu8, Leu71 and Leu73, and BIRC7A’s Arg286 (Fig. 2d,e). To probe the importance of these interactions, we tested the activity of several Ub tail–interacting mutants: UbcH5B’s D87A, I88A and L97A and Ub’s L71D and L73D were defective in BIRC7-mediated Ub transfer (Fig. 2b,c and Supplementary Fig. 2d–g). Furthermore, UbcH5BS I88A~Ub had reduced BIRC7 binding affinity (Table 1), which suggested that the conformation of Ub’s C-terminal tail residues 71–74 is crucial for Ub transfer.

When compared to other UbcH5~Ub structures (PDB 3A33 and 3UGB)24,25, there is a paucity of interactions between the UbcH5 loop, comprising residues 114–119 (loop 114–119), and residues 75 and 76 of Ub. In these other structures, the side chain of Asp117 interacts with the amide of Ub’s Gly76, and loop 114–119 is stabilized by hydrophilic interac-tions with Asn77. In BIRC7239–C–UbcH5BRAS~Ub, the average B factors for the two tail Ub residues (115 Å2) and loop 114–119 (95.7 Å2) are high compared to that of the overall structure; no electron density is present for several of the loop 114–119 side chains, and electron density for the ester linkage is visible but poor around Ub’s Gly75, preventing accurate positioning of the carbonyl group of this residue (Fig. 2e). Nonetheless, modeling of Asn77 from the UbcH5BS~Ub structure (PDB 3A33)24 onto UbcH5BA’s N77A suggests that the asparagine Nd is within hydrogen-bonding distance of UbA’s Gly76 carbonyl oxygen, although the angles may not reflect transition-state geometries (Supplementary Fig. 2h).

Previously, mutational analyses have shown that loop 114–119 is crucial in UbcH5-mediated catalysis4,8,24. Structural alignments of the Ca atoms of UbcH5BRAS~Ub with available structures of UbcH5B and UbcH5B~Ub variants show loop 114–119 adopts multiple conforma-tions (Supplementary Fig. 2i), which suggests that the loop is flexible. Moreover, superposition of UbcH5B from our structure onto the SUMO E2 Ubc9 in the SUMO–RanGAP1–Ubc9–Nup358 complex structure26 (r.m.s. deviation of 1.12 Å for Ca atoms, Supplementary Fig. 2j–l) reveals a similar E2–Ub like protein (Ubl) tail conformation. In Ubc9, Asp127 (corresponding to UbcH5B’s Asp117) contacts the amino group of lysine on the substrate and has been shown to play a critical part in lysine pKa

Figure 1 Structure of BIRC7239–C–UbcH5BRAS~Ub. (a) Cartoon representation of the complex, with the two crystallographic heterotrimers differentiated with A or B subscripts. Top and bottom panels are related by 90° rotation about the x axis. Zn2+ atoms are depicted as gray spheres. Ub, wheat; BIRC7A, green; BIRC7B, yellow; UbcH5B, cyan; covalent linkage, red. UbcH5B a2 and a3 are labeled. (b) Surface representation of the complex, colored and oriented as in a, bottom panel. UbcH5B~Ub linkages and the BIRC7 dimer C-terminal tails are indicated with arrows.

a

BIRC7B BIRC7A

α3

α2

α3

α2

UbcH5BAUbcH5BB

UbA

UbB

90o90o

BIRC7B

BIRC7A

UbcH5B~Ublinkage

α3

α2

α2

α3

α3

α2

α3

α2

UbcH5BA

UbcH5BBUbA

UbB

BIRC7BC-terminal tail

BIRC7AC-terminal tail

b

Table 2 Data collection and refinement statisticsBIRC7239–C–UbcH5B~Ub

Data collection

Space group P43212

Cell dimensions

a, b, c (Å) 100.6, 100.6, 123.9

Resolution (Å) 30–2.18(2.29–2.18)a

Rmerge 0.057(0.621)

I / sI 16.5(3.3)

Completeness (%) 99.8(99.2)

Redundancy 6.7(6.6)

Refinement

Resolution (Å) 30–2.18

No. reflections 33,958

Rwork / Rfree 0.202 / 0.249

No. atoms

Protein 4,232

Ligand/ion 4

Water 153

B factors

Protein 63.9

Ligand/ion 35.1

Water 50.7

R.m.s. deviations

Bond lengths (Å) 0.009

Bond angles (°) 1.24aValues in parentheses are for highest-resolution shell.

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and Fig. 3d), as proposed for RNF4 (ref. 18). To probe our BIRC7A RING–BIRC7B tail–UbcH5BA~UbA arrangement, we hypothesized that a dimer comprising a subunit of BIRC7 with a tail mutation and a second subunit with a RING mutation should be active. For our RING mutation, we generated BIRC7 V254A, which greatly reduces binding affinity for UbcH5BS~Ub and is defective in autoubiqui-tination (Table 1 and Fig. 3e). Incubation of a

mixture of BIRC7 V254A with BIRC7 F296H led to enhanced autou-biquitination activity (Fig. 3e). Thus, the cross-dimer BIRC7A RING–BIRC7B tail arrangement and ensuing interactions with donor Ub are required for UbcH5B~Ub recruitment and Ub transfer.

birc7 binding reorganizes the donor ub conformationOur structural and biochemical data show that BIRC7 promotes Ub transfer by stabilizing UbcH5B and the donor Ub in a conformation optimal for conjugation. To confirm whether the donor Ub forms non-covalent interactions with UbcH5B and BIRC7 in solution, we charged UbcH5BRAS with 15N-labeled Ub (UbcH5BRAS~[15N]Ub) and acquired 1H-15N HSQC spectra in the absence and presence of BIRC7239–C. The UbcH5BRAS~[15N]Ub spectrum showed marked chemical-shift pertur-bations in Ub’s Ile44 patch and C terminus and moderate perturbations in other regions of Ub when compared to the [15N]Ub spectrum alone (Fig. 4a and Supplementary Fig. 4a). As suggested by the shift perturba-tions and the inability of UbcH5BRAS to backside bind, Ub may interact with UbcH5B’s a3 in a similar manner to that observed in Ubc1~Ub and Ube2S~Ub models10,27. Mapping of these perturbations onto UbA reveals that some perturbations radiate away from the UbcH5B–Ub binding interface (Fig. 4b), which suggests that Ub Ile44 patch–UbcH5B interactions are preferred, but donor Ub may populate other conforma-tions, as observed in UbcH5C~Ub complexes14,28.

Titration of an increasing molar ratio of BIRC7239–C to UbcH5BRAS~[15N]Ub produced gradual chemical-shift perturbations in the spectra (Supplementary Fig. 4b–d), indicating changes in the donor Ub interaction network. To separate Ub peak-shift perturbations induced by BIRC7 binding from conjugation of Ub to UbcH5BRAS, we compared the peak shifts from the BIRC7239–C–UbcH5BRAS~[15N]Ub and the UbcH5BRAS~[15N]Ub spectra. These perturbations map to regions near the Leu8-Thr9 loop, Ile36 and Ile44 patches and the C-terminal tail residues Leu71 and Leu73 (Fig. 4c and Supplementary Fig. 4e). These regions correspond to the Ub surfaces involved in nonco-valent interactions with UbcH5B and BIRC7 in our structure (Fig. 4d).

Donor ub ile36 patch–birc7 interactionsIn UbA, the Leu8-Thr9 loop and the patch surrounding Ile36 make exten-sive contacts with the BIRC7A RING domain and BIRC7B’s C-terminal tail (Fig. 3a), providing the first structural evidence, to our knowledge, of donor Ub–RING interactions. UbA’s Leu8, Thr9, Ile36 and Pro37 form hydrophobic contacts with BIRC7A’s Ile284 and Zn2+-binding ligands, His269 and Cys285. In addition, hydrogen bonds occur between the side chains of BIRC7A’s Arg286 and UbA’s Gln40 and between BIRC7A’s His269 and the backbone oxygen of UbA’s Gly35. The C-terminal tail of BIRC7B makes several contacts with UbA: Arg294 forms a hydrogen bond with the backbone oxygen of UbA’s Asp32; Phe296 contacts UbA’s Gly35; and Ser298 sits in a pocket created by UbA’s Thr9, Lys11 and Glu34.

We generated select mutants to probe the observed Ub–BIRC7 inter-actions, including a control Ub mutant, D58A, that opposes the Ile44 and Ile36 patches. BIRC7 Phe296 was mutated to histidine because, for the corresponding residue in RNF4, this mutation did not disrupt dimerization but still considerably reduced activity18. Ub mutants harboring G35E, I36A or Q40R are defective in BIRC7-mediated Ub transfer, whereas Ub D58A had similar activity to wild-type Ub (Fig. 3b and Supplementary Fig. 2f,g). Likewise, BIRC7 I284A, R286A and F296H, which exist as dimers in solution, as shown by size- exclusion chromatography (Supplementary Fig. 3a), are also defective in catalyzing autoubiquitination (Fig. 3c). We introduced V263R or a C-terminal tail deletion (D296–298) to disrupt dimerization. These mutants exist as monomers and are inactive in catalyzing BIRC7 autoubiquitination (Fig. 3c and Supplementary Fig. 3). SPR analyses show that UbcH5BS~Ub G35E and UbcH5BS~Ub I36A have reduced binding affinity for BIRC7, and BIRC7 mutants have reduced binding affinity for UbcH5BS~Ub (Table 1).

The dimeric arrangement shows that E2~Ub can be recruited by both BIRC7 domains, but competent binding of a single UbcH5B~Ub complex requires both BIRC7 domains—the RING domain of one dimer subunit binds UbcH5B and Ub, whereas the tail of the second dimer supports Ub binding through a cross-dimer arrangement (Fig. 1

a b

c

e

d

UbAUbcH5BA Asn41 Wild type

L104A S108R D112A D117A

Wild type

L71D L73D Ub variant

UbcH5B variant

UbcH5B variant~[32P]Ub

Time (s)

Time (s)UbcH5B~Ub variantUbcH5B

0 45 90

0 4590

0 45 90

0 45 90 0 4590

0 45 90 0 45 90 0 4590 0 45 90 0 45 90 0 4590

0 45 90 0 45 90 0 45 90

0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90

L8D R42E I44A G47E Q49E

S22R N77AS22RN77A

H68AV70A

D87A I88A L97A K101E

Cys111

Asp112

Ser108

Leu104

Ser105

Lys101

Leu97

Loop 114–119

Loop 114–119 Loop 114–119

Asp112 Asp117

Ser85

N77A N77A

Leu109

Leu86

Asp87Ile88

Gln92

Leu97

BIRC7AArg286

Lys48

Gln49

α3

Ile44

Arg42His68

Leu69

Lys6

Leu8

Gly75

Arg74Leu73Arg42

Arg72

Leu71Gln40

Leu8

Gly76

Val70

UbcH5BA

UbcH5BA UbcH5BA

UbcH5B~Ublinkage

UbcH5B~Ublinkage

UbA

UbAC-terminal

tail

UbAC-terminal

tail

α2

Figure 2 UbcH5B–Ub interactions within the complex. (a) Close-up view of donor UbA Ile44 patch–UbcH5BA a3 interactions. (b) Nonreduced autoradiograms of pulse-chase reactions showing the disappearance of UbcH5B~[32P]Ub with full-length BIRC7, l-lysine and UbcH5B variants, over time. (c) As in b but for Ub variants and visualized by SDS-PAGE without radiolabeling. (d) Close-up view of UbcH5BA active site–UbA tail interactions. (e) Stereo view of the UbcH5BA~UbA linkage, UbcH5BA loop 114–119 and UbA tail–UbcH5BA interactions, with key residues shown as sticks and labeled. 2Fo – Fc electron density (blue) contoured at 1.0s is shown. For a, d and e, key residues are shown as sticks, and coloring is as in Figure 1, with N atoms blue, O atoms red, and S atoms yellow. Putative hydrogen bonds are shown as dashed lines.

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was used instead of full-length BIRC7. The donor Ub was labeled with [32P]ATP, and we used a histidine-tagged Ub as the accep-tor, which cannot be labeled. At our high-est achievable UbcH5B~Ub concentration (30 mM), our assay only approached satura-tion (Fig. 5a and Supplementary Fig. 5a) and was thus inappropriate for measuring mutational effects on kcat and Km.

Because the BIRC3 RING domain has ~25-fold higher binding affin-ity for UbcH5BS~Ub than does BIRC7 (Table 1), we hypothesized that it might be suitable for kinetic assays to validate our model. The RING domain of BIRC3 resembles BIRC7 (r.m.s. deviation 1.13 Å), and the sequences are 74% identical (Fig. 5b,c). In the sequence alignment, the BIRC7 residues that interact with Ub in our structure are identi-cal to those in BIRC3. All of the BIRC7–Ub interactions observed in our structure are present in a model of a BIRC3–UbcH5B~Ub complex built by superposing the RING domain of BIRC3 onto BIRC7 in our complex (Fig. 5d). Notably, BIRC7 has a valine deletion within a RING

These results suggest that donor Ub has a preference to form noncova-lent interactions with UbcH5B in the absence of E3, but BIRC7 binding further stabilizes the donor Ub interaction network.

Kinetic validation of the birc–e2~ub modelOur data suggest that disruption of E2–Ub or Ub–E3 interactions are expected to hinder binding of E2~Ub and prevent proper posi-tioning of the donor Ub for transfer—under steady-state conditions, an increase in Km and a decrease in kcat are expected for any muta-tion that disrupts these interfaces. We attempted to assess mutational effects on kcat and Km by using kinetic analy-ses on BIRC7-catalyzed diUb formation; to eliminate autoubiquitination, BIRC7239–C

0 2.2 4.5 0 2.2 4.5 0 2.2 4.5 0 2.2 4.5 0 2.2 4.5 0 2.2 4.5 0 2.2 4.5

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Figure 3 Donor Ub–RING interactions are required for BIRC7-catalyzed reactions. (a) Close-up view of donor UbA Ile36 patch–BIRC7 dimer interactions. (b) Nonreduced SDS-PAGE of pulse-chase reactions showing the disappearance of UbcH5B~Ub with full-length BIRC7, l-lysine and Ub variants over time. (c) Nonreduced autoradiograms of pulse-chase reactions showing the simultaneous formation of [32P]Ub products and disappearance of UbcH5B~[32P]Ub with full-length BIRC7 variants over time. Asterisk indicates the E1~Ub band. (d) Experimental design to validate the cross-dimer–donor Ub interactions. BIRC7 mutants expected to disrupt (red Xs) the BIRC7 RING–UbcH5B interaction (V254A) or the BIRC7 tail–Ub interaction (F296H) are shown. A heterodimer of the RING and tail mutants should be more active than a homodimer of either mutant. (e) Reduced autoradiograms showing the formation of [32P]Ub products with wild-type, mutant homodimers and hetero-mutant BIRC7 dimers over time.

0

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a b

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Figure 4 Ub interaction surface in BIRC7239–C–UbcH5BRAS~Ub, identified by NMR. (a) Changes in chemical shift per residue of [15N]Ub, following covalent linkage to UbcH5BRAS, determined by 1H-15N HSQC NMR. Changes were calculated according to the equation [(0.15 dN)2 + dH

2]1/2. (b) Mapping of changes in a (>1s, 0.028 p.p.m.) onto the Ub (red) in the UbcH5BA~UbA portion of BIRC7239–C–UbcH5BRAS~Ub. (c) Changes in chemical shift per residue of Ub in BIRC7239–C–UbcH5BRAS~[15N]Ub compared to UbcH5BRAS~[15N]Ub, calculated as in a. (d) Mapping of changes in c (>1s, 0.01 p.p.m.) onto the Ub (blue) in the BIRC7 dimer–UbcH5BA~UbA portion of BIRC7239–C–UbcH5BRAS~Ub. For a and c, the violet and grey bars represent Gln40 and Gln49 side chain chemical-shift perturbations, respectively. For molecules, coloring is as described in Figure 1.

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thioester linkage for transfer18. The RNF4 study demonstrated that dimerization is required for E3 ligase activity and that both subunits of the dimer are crucial for recruiting E2~Ub, as we observed for BIRC7. In the RNF4–E2~Ub model, the Ile44 surface of Ub was predicted to bind to residues at the dimer interface. Instead, Ub’s Ile44 surface interacts with E2’s a3, and Ub’s Ile36 surface contacts the RING dimer interface to position the donor Ub for transfer.

The UbcH5 family also has the ability to bind Ub’s Ile44 surface via its backside (Ser22 surface). This ability is crucial for the processivity of polyUb chain synthesis14,24, but the mechanism is unknown. Our results and a recent study on cullin-RING ligases9 suggest that the donor Ub cannot interact with the backside of any UbcH5 molecule during trans-fer. Hence, backside binding does not promote polyUb chain formation by positioning the donor Ub for transfer. Although the Ile44 patch on the donor Ub is unavailable during transfer, the backside of the donor UbcH5 (that is, UbcH5~Ub that is bound to E3) is not occupied and may recruit free UbcH5~Ub to enable self-assembly14,24,25 or bind and guide the growing polyUb chain to promote polyUb chain formation14. E2s have been shown to collaborate to catalyze polyUb chain forma-tion29–31, so UbcH5 may also recruit other E2~Ub through backside binding. Further studies are required to elucidate the role of backside binding in polyUb formation.

Optimization of noncovalent donor Ub–E2 interactions and posi-tioning of the thioester linkage are mechanisms shared by other types of E3-catalyzed Ub or Ubl transfers. In the NEDD4L–UbcH5B~Ub complex, the HECT E3 NEDD4L binds both UbcH5B and Ub to posi-tion the E2~Ub thioester linkage for transfer32. The crystal structure of the postconjugated SUMO–RanGAP1–Ubc9–Nup358 complex shows that SUMO E3 Nup358–RanBP2 binds both Ubc9 and SUMO to pre-vent nonproductive Ubc9~SUMO conformations and to position the Ubc9~SUMO thioester linkage for transfer26, though a recent study sug-gests that Ubc9 has only a structural role in this multisubunit E3 com-plex33. For both E3s, the interactions between E3 and donor Ub or Ubl enhance the catalytic efficiency of the transfer reaction by improving kcat and reducing Km. Our SPR analyses of UbcH5BS~Ub variants showed that mutations that disrupt any binding interface increase Kd for the BIRC7–UbcH5B~Ub complex. Using BIRC3541–C, which resembles BIRC7239–C, we demonstrated that disruption of donor Ub–UbcH5B and donor Ub–RING interactions reduces kcat and increases Km for Ub transfer.

RING E3 binding is proposed to induce allosteric changes in E2’s active site to promote Ub transfer8,9. Our data show that conjugation to

hydrophobic patch that is involved in E2 binding, which may account for the observed weaker binding affinities (Fig. 5c and Supplementary Fig. 5b,c). To confirm that the BIRC3 and BIRC7 Ub transfer mecha-nisms are alike, we performed lysine discharge assays using the same sets of mutants tested in the BIRC7 assays. The activity profiles were identi-cal—all of the mutants were defective in Ub transfer except UbcH5B’s S22R and Ub’s D58A (Fig. 5e and Supplementary Fig. 5d,e).

We first measured the kcat and Km for diUb formation by using wild-type Ub, UbcH5B and BIRC3 RING domain to determine whether saturating conditions were achievable. Indeed, the Km was well below the highest achievable UbcH5B~Ub concentration (Fig. 5f). We then selected three mutants for our kinetic assays to validate our model: Ub’s I44A and I36A and BIRC3’s F602H (corresponding to BIRC7’s Phe296). Ub’s Ile44 interacts with Ser108 on UbcH5B’s a3, and Ub’s Ile36 and BIRC3’s Phe602 compose part of the E3–Ub interface. Each of these mutants increased Km and decreased kcat, as predicted by our model (Fig. 5g–i and Supplementary Fig. 5f). Thus, donor Ub interactions with UbcH5B and the RING domain are crucial for optimal Ub transfer.

DiscussionThe BIRC7239–C–UbcH5BRAS~Ub structure and biochemical data presented here provide insights into how a RING E3 and dimerization stabilize the donor Ub to facilitate Ub transfer. Our results agree with a previous study on RNF4, in which modeling and biochemical analyses predicted that the RING E3 binds directly to both E2 and the donor Ub, thereby optimizing the E2~Ub conformation and positioning the

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kcat = 2.4 ± 0.5 min–1

n = 2Km = 32 ± 17 µM

kcat = 0.5 ± 0.2 min–1

n = 4Km = 16.9 ± 1.9 µM

kcat = 1.4 ± 0.1 min–1

UbcH5B (µM) UbcH5B (µM)

UbcH5B (µM) UbcH5B (µM)

Figure 5 Effect of donor Ub interactions on the kinetics of Ub transfer. (a) Single-turnover kinetics of diUb formation catalyzed by BIRC7239–C under pulse-chase conditions. The rate of diUb formation was plotted against UbcH5B concentration. (b) Superposition of the structure of BIRC3541–C dimer (yellow, PDB 3EB6)17 onto BIRC7239–C dimer (green) from BIRC7239–C–UbcH5BRAS~Ub. BIRC3541–C dimer was generated from a symmetry-related molecule. (c) ClustalW sequence alignment of BIRC7 and BIRC3 RING domains. Identical residues are highlighted in yellow and the valine deletion in BIRC7 in red. Cyan and wheat circles indicate residues involved in contacting UbcH5B and Ub, respectively, as observed in BIRC7239–C–UbcH5BRAS~Ub and PDB 3EB6. (d) Models of UbcH5B~Ub bound to BIRC3541–C dimer generated by superposing BIRC3541–C dimer onto BIRC7239–C–UbcH5BRAS~Ub. Key residues involved in donor Ub interactions are indicated. Coloring is as described in Figures 1 and 2. (e) Nonreduced SDS-PAGE of pulse-chase lysine discharge reactions, showing the disappearance of UbcH5B~Ub with variants of BIRC3541–C, UbcH5B or Ub over time. WT, wild type. (f–i) As in a but performed with BIRC3541–C. The rate of diUb formation was plotted against UbcH5B concentration for wild-type reaction (f), [32P]Ub I44A (g), BIRC3541–C F602H (h) and [32P]Ub I36A (i). Kinetic parameters and number of replicates, n, are indicated. Error bars, s.e.m.

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A. Keith for in-house DNA sequencing; and the Diamond Light Source (DLS) for access to beamlines I04 and I24 beamlines (mx6683) that contributed to the results presented here. This work was funded by Cancer Research UK.

AUTHOR CONTRIBUTIONSH.D., L.B. and D.T.H. performed protein purification, assembly of complexes, crystallization and structure determination. H.D. and L.B. conducted and analyzed ubiquitination assays. G.J.S. performed and analyzed SPR experiments. K.C. and G.J.S. performed and analyzed NMR experiments. H.D., L.B. and D.T.H. wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://www.nature.com/doifinder/10.1038/nsmb.2397.Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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Ub and binding to an E3 do not substantially alter E2’s active site as com-pared to other structures of free UbcH5, UbcH5~Ub and UbcH5–E3. There is a slight shift in loop 114–119 of the E2, but whether this is due to E3 binding, our Asn77 mutation or inherent flexibility is unclear. Our results support findings from previous mutational analyses, in which UbcH5’s a2 was revealed to contribute to the allosteric communica-tion induced by E3 binding, possibly through interactions with Ub: UbcH5B I88A reduced and UbcH5C D87E abolished E3-stimulated Ub transfer4,8. In our complex, Ub tail residues 71–74 interact with a2 of UbcH5B. BIRC7’s Arg286, which is strictly conserved in many RING E3s as arginine or lysine, not only binds UbcH5B’s a2 in our structure but also coordinates Ub’s tail residues 71–74 to a2 of the E2.

The cross-dimer arrangement observed here and in RNF4 (ref. 18) is required for donor Ub interactions and E2~Ub recruitment. We find that, when compared to other dimeric RING E3s that function with the UbcH5 family E2s, this dimer arrangement is also observed in the struc-tures of MDM2–MDMX34 and IDOL35 but is absent in the structures of BRCA1–BARD1 (ref. 36) and RNF2–BMI1 (refs. 19,37). Modeling of the UbcH5BRAS~Ub fragment of our structure onto MDM2–MDMX reveals the presence of a complementary donor Ub–RING interface and a conserved phenylalanine (Phe296 in BIRC7), on the C-terminal tail, required for cross-dimer donor Ub binding (Supplementary Fig. 5g). In contrast, a similar model of RNF2–BMI1 reveals potential side chain clashes and the absence of favorable cross-dimer donor Ub interactions (Supplementary Fig. 5h). Indeed, our data (Table 1) and previous stud-ies18,21 show that the BIRCs, MDM2–MDMX and RNF4 have binding preferences for E2~Ub rather than for free E2, whereas RNF2–BMI1 has no preference. Clearly, not all dimeric RING E3s recruit E2~Ub in a similar fashion, but our results suggest that RING E3s that adopt con-formations similar to the BIRC7 RING dimer may have similar mecha-nisms of E2~Ub recruitment and Ub transfer.

This mechanism may not be unique to dimeric RING E3s. U-box domains structurally resemble RING domains, and some form active homodimers in a manner analogous to RING E3 dimerization. The struc-ture of PRP19 U-box dimer shows a similar U-box C-terminal tail con-figuration to BIRC7 but with variations in the C-terminal tail sequence38. Modeling of the UbcH5BRAS~Ub portion of our structure onto the PRP19 U-box structure reveals a complementary interface between donor Ub and U-box, with slight differences in the mode of C-terminal tail interactions (Supplementary Fig. 5i), which suggests a similar E2~Ub recruitment mechanism. Interestingly, several monomeric RING domains also exhibit higher binding affinities for E2~Ub than E2, despite lacking the cross-dimer arrangement. Examples include cullin-RING ligases39, E3a (ref. 40) and c-CBL41. Common UbcH5B surface residues outside the E2’s active site and E3 binding site, for example Ile88, are essential for Ub transfer mediated by Apc2–Apc11 and CNOT4 (ref. 8). We speculate, on the basis of these observations, that other RING E3s may use similar modes of E2~Ub recruitment and Ub transfer. Finally, our results are supported by recent structural findings on the RNF4 RING–UbcH5A~Ub complex42.

metHoDsMethods and any associated references are available in the online ver-sion of the paper.

Accession codes. Protein Data Bank: Coordinates and structure factors have been deposited with accession code 4AUQ.

Note: Supplementary information is available in the online version of the paper.

ACKNOWLEDGMENTSWe would like to thank K. Vousden and A. Schuettelkopf for discussion. We thank A. Krishnan, a former lab technician, for purification of BIRC3541–C; W. Clark and

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doi:10.1038/nsmb.2379 nature structural & molecular biology

242–298 and chain E residues 242–298), Ub (chain C residues 1–76 and chain F residues 2–76). Residues with poor side chain electron density were built as ala-nine. Details of the refinement statistics are shown in Table 2. All figure models were generated using PyMOL (Schrödinger).

In vitro pulse-chase assays. E2 (10 ml) was charged in a buffer containing 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 5 mM ATP, 1 mM DTT, 0.3 U ml–1 inorganic pyrophosphatase, 0.3 U ml–1 creatine kinase, 5 mM creatine phosphate, 1 mg ml–1 BSA (except for assays detected with SimplyBlue SafeStain (Invitrogen)) and typically containing mouse Uba1 (0.4 mM), Ub or [32P]Ub (40 mM) and UbcH5B (20 mM) for 15–30 min at 23 °C. Charging was stopped by incubating the reaction with 0.25 U apyrase (Sigma) and 30 mM EDTA for 5 min at 23 °C. The autoubiquitination, SMAC ubiquitination, diUb and lysine discharge reac-tions were then respectively initiated by the addition of BIRC7 variants (2 mM), a mixture of BIRC7 (0.220 mM) and [32P]SMAC (5.6 mM), a mixture of BIRC7 (0.220 mM) and His-Ub (200 mM), a mixture of BIRC7 (2 mM) and l-lysine (150 mM) or a mixture of BIRC3541–C (0.220 mM) and l-lysine (150 mM). The parentheses indicate the final concentration. The final UbcH5B~Ub concentration was ~7 mM for all reactions. Reactions were quenched with 2× SDS loading buffer (with DTT only for the diUb assays) at the indicated time, resolved by SDS-PAGE, dried and visualized by autoradiography, stained with SimplyBlue SafeStain or immunoblot-ted with Ub (1:2,000 dilution, clone P4D1-A11, 05-944, Millipore) or His-probe (1:500 dilution, clone H-15, sc-803, Santa Cruz) antibodies.

Single-turnover kinetics of diUb formation. UbcH5B was charged in a buffer containing 50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 5 mM ATP, 1 mM DTT, 0.3 U ml–1 inorganic pyrophosphatase, 0.3 U ml–1 creatine kinase, 5 mM creatine phosphate, 2.5 mM mouse Uba1, 130 mM [32P]Ub variant and 66 mM UbcH5B for 15 min at 23 °C. Charging was stopped as described above. Different amounts of UbcH5B~[32P]Ub variants were then added to a reaction containing 50 mM HEPES, pH 7.5, 50 mM NaCl, 820 mM His-Ub (final concentration) and BIRC7239–C or BIRC3541–C variants. Final E3 concentrations are indicated in Supplementary Figure 5a,f. Reactions were quenched at 30 s with 2× SDS load-ing buffer containing 200 mM DTT, resolved by SDS-PAGE, dried and exposed to a phosphorimager. Under these conditions, less than 15% of the UbcH5B~[32P]Ub variants were transferred to His-Ub, thus representing the initial rate. In addition, there was no observable autoubiquitination of BIRC3541–C variants and BIRC7239–C. DiUb bands were quantified using ImageQuant (GE Healthcare). Control reactions lacking E3 at each UbcH5B~[32P]Ub variant concentration were performed for background subtraction during quantification. All reported kinetic parameters were determined by fitting at least two independent data sets to the Michaelis-Menten equation, using SigmaPlot 8.0 (Systat Software Inc.).

E3–E2~Ub binding assays. GST-E3s were coupled to CM-5 chips (Biacore Life Sciences) as described previously41. All chips were equilibrated in running buffer containing 25 mM Tris, pH 7.6, 150 mM NaCl, 0.1 mg ml–1 BSA, 1 mM DTT and 0.005% (v/v) Tween-20, and analytes were serially diluted in running buffer. Binding was measured at concentration ranges of 0-180 mM for analytes. Data reported are the difference in SPR signal between GST-E3 variants and GST alone. The data were analyzed by steady-state affinity analysis using the Biacore T200 Evaluation software package (Biacore Life Sciences) and Scrubber2.0c (BioLogic Software).

NMR spectroscopy. Data were recorded at 25 °C on a Varian VNMRS 600 spec-trometer using a 5-mm triple-resonance (HCN) cold probe. All protein samples were prepared or dialyzed in 25 mM sodium phosphate, 0.15 M NaCl buffer at pH 7.0, containing 90/10% H2O/D2O. 1H-15N HSQC spectra were recorded for [15N]Ub (100 mM) and UbcH5BRAS~[15N]Ub (100 mM). For BIRC7239–C titration experiments, the 1H-15N HSQC spectra of BIRC7239–C–UbcH5BRAS~[15N]Ub complex were recorded with a molar ratio of BIRC7239–C:UbcH5BRAS~[15N]Ub at 100:100 mM and 300:200 mM. Ub chemical shifts are available in BioMagResBank under accession code 6457. Owing to large chemical-shift changes upon cova-lent linkage to UbcH5BRAS and resonance broadening, Gln31, Arg42, Val70 and Arg74 were not assigned in Figure 4 and Supplementary Figure 4. The chem-ical-shift perturbations were estimated using the equation [(0.15 dN)2 + dH

2]1/2 (p.p.m.), where dN and dH represent changes in nitrogen and proton chemical shifts, respectively, and analyzed using CCPN50. An iterative procedure was used to determine 1s by calculating a corrected s.d. to zero as described previously51 but was stopped after 10% of data were excluded.

online metHoDsProtein preparation. Constructs were generated by PCR ligation techniques and sequences verified by automated sequencing. All proteins are from human unless specified. BIRC3541–C variants, BIRC7239–C, full-length BIRC7 variants, RNF21–114 and MDM2428–C were cloned into pGEX4T1 (GE Healthcare), which contains an N-terminal glutathione S-transferase (GST) tag followed by a TEV protease cleavage site. BMI11–109 and MDMX428–C were cloned into pRSF_DUET (Novagen), which contains an N-terminal His tag followed by a TEV protease cleavage site. Untagged UbcH5B variants were cloned into pRSF_1b vector. Proteins were expressed in E. coli BL21 (DE3) Gold cells. For crystallization, BIRC7239–C was purified by glutathione affinity chromatography, treated with TEV protease to cleave the GST-tag, and purified by desalting/glutathione-affinity pass-back and size-exclusion chromatography. For Biacore SPR analyses, GST-tagged BIRC3541–C, BIRC7239–C and full-length BIRC7 variants were purified by glutathione affinity; GST-MDM2428–C–His-MDMX428–C and GST-RNF21–114–His-BMI11–109 were purified by Ni-NTA chromatography followed by glutathi-one affinity chromatography; UbcH5B variants were purified by cation-exchange and size-exclusion chromatography. For ubiquitination assays, mouse Uba1 was expressed from pET23d (Novagen), charged with GST-Ub with 5 mM MgCl2 and 5 mM ATP for 2 h at 4 °C and purified by glutathione affinity chromatography with elution by 20 mM DTT, followed by anion exchange chromatography; [32P]Ub variants expressed from pGEX2TK and His-Ub were prepared as described previ-ously41. BIRC3 and BIRC7 variants were purified as described above, except cleav-age was performed on glutathione Sepharose beads and the pass-back step omitted. [32P]SMAC comprising residues 56–C was expressed from pET23d containing a C-terminal protein kinase A recognition sequence (RRAVS) and purified by Ni-NTA affinity chromatography followed by size exclusion chromatography. All protein concentrations were determined by Bradford assay43 with bovine serum albumin (BSA) as a standard, and Ub concentration was determined as described previously44. Proteins were stored in 25 mM Tris-HCl, pH 7.6, 0.15 M NaCl and 1 mM DTT or 25 mM HEPES, pH 7.0, 0.15 M NaCl and 1 mM DTT at –80 °C.

Generation of UbcH5BS~Ub variants. Ub variants with a C-terminal Gly-Gly motif were cloned into pGEX4T1 to add an N-terminal GST-tag followed by a TEV cleavage site and a Gly-Gly-Ser linker. GST-Ub variants were purified by glutathione affinity chromatography. Untagged Arabidopsis thaliana Uba1 was expressed from pET23d and purified as described above for mouse Uba1. Untagged UbcH5B C85S variants were expressed from pRSF_1b and purified by cation-exchange chromatography. All proteins were dialyzed into 25 mM HEPES, pH 8.0, 0.15 M NaCl. UbcH5BS~Ub variants were formed by mixing Uba1, UbcH5B C85S variants, GST-Ub variants and TEV protease in the pres-ence of 10 mM MgCl2 and 10 mM ATP at 19 °C for 1 d. UbcH5BS~Ub variants were then purified by cation-exchange chromatography followed by gel-filtration chromatography using a Hi-Load 16/60 SD75 column. All UbcH5BS~Ub variants were stored in 25 mM HEPES, pH 7.5, 0.2 M NaCl and 1 mM DTT. UbcH5BS~Ub variant concentrations were determined by measuring absorbance at 280 nm in the presence of 6 M guanidine HCl and using calculated molar extinction coef-ficient (26,900 M-1 cm-1) for Biacore SPR analyses.

Crystallization. Crystals were obtained by mixing the protein with an equal vol-ume of reservoir solution and were grown by hanging-drop vapor diffusion at 4 °C. BIRC7239–C (4.7 mg ml–1) was mixed with UbcH5BRAS~Ub (25.4 mg ml–1) at a 1:1 molar ratio, and crystals were grown in conditions containing 100 mM HEPES, pH 7.0, 17-20% (w/v) PEG 3350 and 0.2 M triammonium citrate. The crystals were flash frozen in 100 mM HEPES, pH 7.0, 23% (w/v) PEG 3350, 0.2 M triammonium citrate and 20% (v/v) glycerol. Data were collected at beamlines I04 and I24 at DLS.

Structural determination. The data were integrated with automated XDS45 and scaled using the CCP4 program suite46. BIRC7239–C –UbcH5BRAS~Ub complex crystals belong to space group P43212 with two molecules in the asymmetric unit. Initial phases of BIRC7239–C–UbcH5BRAS~Ub were obtained by molecular replacement with PHASER47, using individual domains from PDB 3EB6 and PDB 3A33 as the search models. All models were built in COOT48 and refined using PHENIX49.

The structure of BIRC7239–C–UbcH5BRAS~Ub (chains A–F) was refined at a resolution of 2.18 Å, and the final model contained two copies of UbcH5B (chain A residues 2–147 and chain D residues 2–147), BIRC7239–C (chain B residues

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software pipeline. Proteins 59, 687–696 (2005).51. Schumann, F.H. et al. Combined chemical shift changes and amino acid specific chemi-

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44. Bohnsack, R.N. & Haas, A.L. Conservation in the mechanism of Nedd8 activation by the human AppBp1-Uba3 heterodimer. J. Biol. Chem. 278, 26823–26830 (2003).

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