Article
Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin Graphical Abstract
Authors Lori Buetow, Mads Gabrielsen, ..., Brian O. Smith, Danny T. Huang
Correspondence [email protected]
In Brief Buetow et al. show that non-covalent ubiquitin binding to the backside of E2 stimulates RING E3-catalyzed initial ubiquitin transfer and poly-ubiquitin chain formation. Crystal structures and biochemical analyses reveal that backside bound ubiquitin stabilizes RING E3-E2ubiquitin complex for catalysis.
Highlights B
Accession Numbers D
d
Ub activates RING E3-UbcH5BUb complex for Ub transfer
d
Free Ub, E2Ub, and monoUb and polyUb chains on E3 and substrate act as UbB sources
d
d
UbB enhances RING E3-UbcH5B–Ub affinity, and RING E3UbcH5B–Ub improves UbB affinity Structure-based insights into UbB stimulatory mechanism
Buetow et al., 2015, Molecular Cell 58, 1–14 April 16, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.02.017
4V3K 4V3L
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Molecular Cell
Article Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin Lori Buetow,1 Mads Gabrielsen,1 Nahoum G. Anthony,2 Hao Dou,1,4 Amrita Patel,1 Hazel Aitkenhead,1 Gary J. Sibbet,1 Brian O. Smith,3 and Danny T. Huang1,* 1Cancer
Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK 3Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 4Present address: Institute of Medical Genetics, School of Medicine, Shandong University, No. 44 Wenhuaxi Road, Jinan, Shandong 250012, People’s Republic of China *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.02.017 2Strathclyde
SUMMARY
RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (UbB) enhances both RING-independent and RINGdependent UbcH5B-catalyzed donor Ub (UbD) transfer, but with a more prominent effect in RING-dependent transfer. UbB enhances RING E3s’ affinities for UbcH5B–Ub, and RING E3-UbcH5B–Ub complex improves UbB’s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B–Ub in the absence and presence of UbB, together with molecular dynamics simulation and biochemical analyses, suggests UbB restricts the flexibility of UbcH5B’s a1 and a1b1 loop. UbB supports E3 function by stabilizing the RING E3UbcH5B–Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, UbB serves as an allosteric activator of RING E3-mediated Ub transfer.
INTRODUCTION Post-translational modification by Ub plays a crucial role in regulating a plethora of events in eukaryotic cells by altering proteins’ half-lives, interacting partners, and activities. Ub modification is achieved by sequential actions of Ub-activating enzyme (E1), E2, and E3. E1 activates and transfers Ub’s C terminus to E2’s catalytic cysteine, forming an E2Ub thioester intermediate ( indicates a thioester). E3 recruits E2Ub and substrate to facilitate Ub transfer from E2 to a substrate lysine side chain or N-terminal methionine. There are four classes of E3s: RING, HECT, U-box, and RING-in-between-RING that utilize varying mechanisms to transfer Ub to substrate (Dye and Schulman, 2007; Spratt
et al., 2014). RING E3s comprise the largest family of E3s and function by recruiting E2Ub via the RING domain and substrate(s) via protein-protein interaction domain(s) to promote direct Ub transfer from E2 to substrate. RING E3s lack active sites, instead functioning as scaffolds. Biochemical and recent structural studies have shown that RING domains bind E2Ub, restraining UbD in a closed configuration where numerous RING-UbD and E2-UbD interactions optimally position UbD’s C-terminal tail within E2’s active site cleft and orient the thioester for nucleophilic attack (Dou et al., 2012; Plechanovova´ et al., 2012; Pruneda et al., 2012; Saha et al., 2011; Wickliffe et al., 2011). Although the RING domain is sufficient to populate E2Ub in the closed active configuration, an additional component outside the RING domain helps maintain this state and enhances the catalytic efficiency of Ub transfer (Dou et al., 2013; Plechanovova´ et al., 2012). Examples include the phosphorylated linker tyrosine in Cbl (Dou et al., 2013) and the C-terminal tail of RING E3 dimers (Dou et al., 2012; Plechanovova´ et al., 2012). Residues aligning the E2 active site and the chemical environment of the acceptor amine also influence RING E3-catalyzed Ub transfer (Berndsen et al., 2013; Ozkan et al., 2005; Scott et al., 2014; Wickliffe et al., 2011; Wu et al., 2003; Yunus and Lima, 2006). Furthermore, some RING E3s such as gp78, Cue1p, and Rad18 harbor additional E2-binding motifs that bind the backside of Ube2g2, Ubc7p, and Rad6, respectively (Das et al., 2009, 2013; Hibbert et al., 2011; Li et al., 2009; Metzger et al., 2013). In gp78 and Cue1p, the additional E2-binding element modulates RING E3-E2 binding affinity to facilitate Ub transfer, whereas the additional E2-binding motif in Rad18 serves to direct monoubiquitination. Along with E2-binding motifs on select E3s, Ub has also been shown to bind to the backside of several E2s, including Rad6, UbcH5, UbcH6, and Ube2g2 families (Bocik et al., 2011; Brzovic et al., 2006; Hibbert et al., 2011; Miura et al., 1999). This backside interaction is important for processivity in polyUb chain formation and Ub transfer to substrate (Brzovic et al., 2006; Ranaweera and Yang, 2013). For the UbcH5 family, UbB is proposed to facilitate processivity by forming self-assembled oligomers of UbcH5Ub, thereby increasing local concentrations of substrate and/or by bridging the gap between the E2- and substrate-binding domains of the E3 (Brzovic et al., 2006; Page Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 1
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Figure 1. UbB Stimulates RING-Dependent and RING-Independent Ub Transfer (A) Reduced autoradiogram showing autoubiquitination by GST-RNF38-RING with 32P-Ub and UbcH5B variants over time. The asterisk (*) represents nonreducible Ub-E1. (B) As in (A) but for full-length BIRC4 instead of GST-RNF38-RING. (C) Reduced autoradiogram showing ubiquitination of SMAC-32P by BIRC4 and UbcH5B variants over time. (D) Reduced autoradiograms showing diUb formation over time by UbcH5B variants with (top) or without RNF38-RING (bottom). Different exposure times were used for the panels. (E) Kinetics of diUb formation by RNF38-RING with varying concentrations of WT and UbcH5B S22R. Three rate replicates were measured for each UbcH5B concentration. Kinetic parameters and 95% CI are indicated. Error bars represent SD. (legend continued on next page)
2 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
et al., 2012; Sakata et al., 2010). In addition, polyUb chains on E3s purportedly serve as scaffolds for backside binding UbcH5Ub to mediate RING-independent Ub transfer to substrate (Ranaweera and Yang, 2013). Which of these mechanisms predominates, if any, is unknown and requires further investigation. Structures of UbcH5 bound non-covalently to Ub reveal that Ub’s Ile44 surface interacts with UbcH5’s b1-3 surface centering on Ser22 (Brzovic et al., 2006). Disruption of either surface hinders RING E3-catalyzed polyUb chain formation but the effect on backside-mediated Ub transfer has not been thoroughly characterized. Here, we uncover an original role for UbB in promoting ubiquitination. We show that UbB directly simulates RING-independent and RING-dependent Ub transfer. Using the RING domain of RNF38, an E3 ligase implicated in p53 ubiquitination (Sheren and Kassenbrock, 2013), as a model system, we determine crystal structures of RNF38-RING bound to UbcH5B–Ub alone and in complex with free Ub bound to UbcH5B’s backside. Comparison of the structures, together with other biophysical and biochemical analyses, reveals that UbB alters UbcH5B dynamics and transforms RNF38-UbcH5B–Ub into a higher affinity complex with improved catalytic efficiency. Our results demonstrate UbB functions as an allosteric activator of RING E3-E2Ub complexes to enhance Ub transfer. RESULTS UbB-UbcH5B Interaction Stimulates RING-Independent and RING-Dependent Ub Transfer For the UbcH5 family of E2s, S22R substitution abrogates UbB interactions and is defective in RING E3-catalyzed polyUb chain formation (Brzovic et al., 2006). To validate these findings, we performed ubiquitination assays with wild-type UbcH5B (WT) and UbcH5B S22R on N-terminally glutathione-S-transferase tagged RNF38 RING domain (GST-RNF38-RING), full-length BIRC4 and the BIRC4 substrate SMAC. Polyubiquitination or multimonoubiquitination of GST-RNF38-RING, BIRC4, and SMAC were diminished with UbcH5B S22R compared with WT (Figures 1A–1C) but not essentially eliminated as observed in the BRCA1 autoubiquitination assays (Brzovic et al., 2006). During Ub transfer, RING E3 binds E2Ub, optimizes UbD in the closed active configuration, and positions substrate or acceptor Ub (UbA) for catalysis. To assess the effects of UbB on Ub transfer, we performed in vitro single turnover diUb formation assays using WT and UbcH5B S22R; with surface plasmon resonance (SPR), we estimated a KD of 200 mM for the UbBUbcH5B interaction (Figure S1; Table 1), similar to the reported KD for the UbB-UbcH5C interaction (300 mM; Brzovic et al.,
2006). UbcH5B variants were precharged with equimolar concentrations of 32P-Ub and then chased by adding saturated Ub lacking the C-terminal Gly-Gly motif (UbDGG), which cannot be charged by Ub E1. Surprisingly, UbcH5B S22R was slower in diUb formation whether E3 was present or not (Figure 1D), but the overall effects were more obvious with E3 (Figure S2A). We then measured the kinetic constants of diUb formation with RNF38-RING and found Km decreased 8-fold, while kcat increased 2-fold for WT compared with UbcH5B S22R (Figures 1E and S2B). To exclude potential UbB mediated effects on stabilizing or recruiting UbA within the E-S intermediate complex, we tested single-turnover Ub transfer to lysine in pulse chase assays with and without 300 mM K0UbDGG, where all seven lysines were mutated to arginine to prevent Ub acting as an acceptor. In the absence of K0UbDGG, regardless of whether RNF38-RING was present, the rates of UbcH5BUb and UbcH5B S22RUb discharge were comparable, whereas including K0UbDGG increased the rate of discharge for UbcH5BUb but not for UbcH5B S22RUb (Figure 1F). Because of the quick rate of discharge and large variability, quantification of stimulation was not possible. Although lysine concentrations differed between assays with and without RNF38-RING (10 mM and 150 mM, respectively), effects were more obvious when E3 was present. To test the specificity of the UbB-UbcH5B interaction, we then performed our lysine discharge assays with K0UbDGG I44A since this mutation also abrogates the UbBUbcH5B interaction. K0UbDGG I44A did not promote UbcH5BUb discharge under any condition tested, and the rate of discharge was similar to assays performed with UbcH5B S22RUb, suggesting that both UbcH5B S22R and K0UbDGG I44A exclusively impact backside-mediated stimulation (Figure 1F). Furthermore, we also tested a dimeric RING E3, BIRC4, and a U-box E3, UBE4B, and found that K0UbDGG promoted UbcH5BUb discharge in both E3-mediated reactions (Figure 1G), establishing that this feature is not exclusive to RNF38-RING. Together, these data demonstrate that UbB plays a distinct role in stimulating Ub transfer unrelated to acting as a donor or acceptor. This effect occurs in a RING-dependent and RING-independent manner, but is more pronounced in the presence of a RING domain. UbB functions to stabilize the E-S intermediate complex when RNF38-RING is present, suggesting that the binding affinity between the RING domain and UbcH5BUb might be enhanced. UbB-RING E3 Synergy Enhances UbcH5B–Ub Binding Interactions To investigate whether UbB improves the binding affinity of RNF38-RING for UbcH5BUb, we performed SPR analyses to
(F) Non-reduced autoradiograms of pulsed-chased reactions showing the disappearance of UbcH5B32P-Ub variants over time with lysine in the presence and absence of K0UbDGG or K0UbDGG I44A, as indicated with RNF38-RING (top) or without E3 (bottom). (G) As for (F) but with UbcH5B32P-Ub, K0UbDGG, the RING domains of RNF38 (top) and BIRC4 (middle) and UBE4B U-box (bottom). (H) Non-reduced antibiotin immunoblots of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5BUbC over time with K0UbDGG and RNF38-RING as indicated. (I) Non-reduced autoradiograms of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5B32P-Ub variants over time with (left) or without RNF38-RING (right) in the presence and absence of K0UbDGG. See also Figure S2.
Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 3
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Table 1. KD for Interactions between RING E3 Variants, UbcH5B Variants, UbcH5B–Ub Variants, and Ub Immobilized Protein
Analytea
KD (mM)
Binding Enhancement (Fold)b
GST-RING E3 RNF38-RING
UbcH5B
89 ± 1
RNF38-RING
UbcH5B + 600 mM UbDGG
73 ± 2
RNF38-RING
UbcH5B–Ub
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BS22R–Ub
RNF38-RING
UbcH5BS22R–Ub + 600 mM UbDGG
1.3 ± 0.1
RNF38-RING
UbcH5B–Ub + 3 mM UbDGG
0.5 ± 0.01
RNF38-RING
UbcH5B–Ub + 10 mM UbDGG
0.33 ± 0.01
RNF38-RING
UbcH5B–Ub + 30 mM UbDGG
0.22 ± 0.01
RNF38-RING
UbcH5B–Ub + 100 mM UbDGG
0.11 ± 0.01
RNF38-RING
UbcH5BE9D–Ub
0.37 ± 0.04
RNF38-RING
UbcH5BE9D–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BQ20A–Ub
RNF38-RING
UbcH5BQ20A–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BI37A–Ub
RNF38-RING
UbcH5BI37A–Ub + 600 mM UbDGG
0.79 ± 0.05
4.6
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG L8A
0.13 ± 0.02
7.1
RNF38-RING
UbcH5B–Ub + 100 mM UbcH5BS22R,F62A,P95D–Ub
0.20 ± 0.01
RNF38-RING
UbcH5BS22R–Ub + 100 mM UbcH5BS22R,F62A,P95D–Ub
0.94 ± 0.02
1.2
0.92 ± 0.08
C85S
–Ub
0.077 ± 0.006
11.9
1.04 ± 0.12
0.046 ± 0.003
0.8
8.0
2.2 ± 0.4 0.26 ± 0.02
8.5
3.6 ± 0.6
4.6 ± 0.2
RNF38-RING
UbcH5B
RNF38-RING
UbcH5BC85S–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BC85S,I88A–Ub
27 ± 1
RNF38-RING
UbcH5BC85S,I88A–Ub + 600 mM UbDGG
4.8 ± 0.1
RNF38-RING
UbcH5BC85S,S108R–Ub
74 ± 2
RNF38-RING
UbcH5BC85S,S108R–Ub + 600 mM UbDGG
BIRC4-RING
UbcH5B–Ub
BIRC4-RING
UbcH5B–Ub + 600 mM UbDGG
BIRC4-RING
UbcH5BS22R–Ub
33.8 ± 2.4
BIRC4-RING
UbcH5BS22R–Ub + 600 mM UbDGG
36.5 ± 2.4
UBE4B-U-box
UbcH5B–Ub
29 ± 0.5
UBE4B-U-box
UbcH5B–Ub + 600 mM UbDGG
4.9 ± 0.3
0.36 ± 0.01
76 ± 2
12.8 5.6 1.0
39.1 ± 1.3
S22R
6.2 ± 0.6
UBE4B-U-box
UbcH5B
UBE4B-U-box
UbcH5BS22R–Ub + 600 mM UbDGG
44.8 ± 1.4
Ub
UbcH5B
206 ± 6
Ub
UbcH5B–Ub
284 ± 8
Ub
UbcH5B–Ub + molar excess of RNF38-RING
Ub
RNF38-RING
Ub
UbcH5B Q20A
197 ± 17
Ub L8A
UbcH5B
296 ± 7
Ub L8A
UbcH5B–Ub
353 ± 7
Ub L8A
UbcH5B–Ub + molar excess of RNF38-RING
–Ub
6.3 0.92 5.9
44.2 ± 1.7 0.99
GST-Ub
14 ± 1.8
20
no binding
44 ± 3
8
MBP-tag RNF38-RING
UbcH5B–Ub
2.04 ± 0.05
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG
0.08 ± 0.007
26 (Continued on next page)
4 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Table 1.
Continued
Immobilized Protein
Analytea
Ub
UbcH5B–Ub
137 ± 11
Ub
UbcH5B–Ub + molar excess of RNF38-RING
4.9 ± 1.4
KD (mM)
Binding Enhancement (Fold)b 28
SEM is indicated. Number of replicates, representative sensorgrams, and binding curves are shown in Figure S1. a Analytes containing fixed concentration of UbDGG variants, UbcH5BS22R,F62A,P95D–Ub or RNF38-RING are indicated. b The degree of binding enhancement in the presence of UbDGG variant or RNF38-RING was determined by using the KD in the absence of UbDGG variant or RNF38-RING divided by the KD in the presence of UbDGG variant or RNF38-RING, respectively.
measure the binding affinity of GST-RNF38-RING for stably linked UbcH5B–Ub alone and with 600 mM UbDGG. We generated stable conjugates by mutating UbcH5B’s catalytic cysteine to a lysine or serine, thereby mimicking UbcH5BUb by forming, respectively, a stable isopeptide (UbcH5B–Ub) or oxyester (UbcH5BC85S–Ub) linkage with Ub. Though UbcH5B– Ub binds slightly tighter than UbcH5BC85S–Ub, in both instances, UbDGG improved binding affinity by 12-fold (Table 1). We then measured GST-RNF38-RING’s binding affinity for UbcH5BS22R– Ub and found no improvement upon including UbDGG (Table 1). To ensure that GST dimerization did not contribute to binding enhancement, we determined the binding affinity of MBPRNF38-RING for UbcH5B–Ub under similar conditions by SPR and found UbDGG enhanced binding by 26-fold (Table 1). When we performed the same experiments with GST-BIRC4RING and GST-UBE4B-U-box, UbDGG improved binding affinities for UbcH5B–Ub by 6-fold but not for UbcH5BS22R–Ub (Table 1). Thus UbB enhances RING/U-box E3-UbcH5B–Ub interactions but the extent of enhancement depends on the complex. Interestingly, when the same experiments were performed using UbcH5B lacking conjugated Ub, addition of UbDGG only improved RNF38-RING’s binding affinity for UbcH5B by 1.2fold (Table 1), suggesting that the presence of UbD is required for UbB-mediated binding enhancement. Since UbB improved RNF38’s binding affinity for UbcH5B–Ub, we asked whether RNF38 affects UbB-UbcH5B–Ub binding affinity. We performed SPR analyses to determine GST-RNF38RING’s binding affinity for UbcH5B–Ub with varying UbDGG concentrations. Addition of 3 mM UbDGG marginally increased RNF38-RING’s binding affinity for UbcH5B–Ub, and this binding affinity progressively improved with increasing UbDGG concentrations, approaching saturation at 100 mM UbDGG (Table 1). Consistent with the SPR results, the rate of RNF38-mediated UbcH5BUb lysine discharge with increasing K0UbDGG concentrations approached saturation near 100 mM K0UbDGG (Figure S2C). To determine the KD of UbB for RNF38-UbcH5B–Ub, we used SPR to measure GST-Ub’s binding affinity for UbcH5B–Ub alone and with saturated RNF38-RING. Without RNF38-RING, GST-Ub displayed weak binding affinities for UbcH5B–Ub (284 mM) and UbcH5B (206 mM); remarkably, RNF38-RING enhanced GST-Ub’s binding affinity for UbcH5B–Ub by 20fold; similarly, the addition of UbDGG enhanced the binding affinity of MBP-Ub for UbcH5B–Ub by 28-fold (Table 1). Activation of RING E3-UbcH5BUb by UbB RING E3s lock E2Ub into a closed conformation (Dou et al., 2012; Plechanovova´ et al., 2012; Pruneda et al., 2012) and
induce global changes in E2 (Benirschke et al., 2010) to activate the E2Ub thioester bond for catalysis. We questioned how UbB influences this mechanism. The globular body of UbD contributes the bulk of UbD interactions with UbcH5B and RING E3 and is required for E3-induced active site conformational changes, so we initially investigated whether the globular body of UbD is also important for UbB stimulation. We performed lysine discharge assays on UbcH5B charged with a biotin-labeled Ub C-terminal peptide (residues 71–76; UbC) lacking the globular Ub body. Discharge of UbcH5BUbC was not mediated by RNF38-RING or stimulated by K0UbDGG (Figure 1H). Thus, the globular body of UbD is required for RING E3-mediated catalysis and UbB-stimulated Ub transfer. Next we examined UbcH5B mutants with well-characterized defects in E3-mediated Ub transfer. We used UbcH5B I88A (an a2 residue that contacts UbD’s tail), S108R (an a3 residue that contacts UbD’s Ile44 surface), and I37A (Ile37 is on the b sheet abutting the UbcH5B a3-UbD Ile44 interaction surface) substitutions in our lysine discharge assays. As a control, we included UbcH5B E140A; Glu140 is a surface residue and does not contact any interacting partners in available E3-E2–Ub complexes. UbcH5B E140A behaved comparably to WT UbcH5B in all conditions. K0UbDGG did not stimulate discharge of any mutant in the absence of E3. With RNF38-RING, although Ub discharge was impaired by UbcH5B I88A and I37A, addition of K0UbDGG still had a stimulatory effect, whereas UbcH5B S108R abolished both E3-mediation and K0UbDGG-stimulation of Ub transfer (Figures 1I and S2D). Whether UbcH5B I37A and I88A simply impaired E3-facilitated discharge or also caused defects in UbB-mediated stimulation was unclear, so we generated a stably linked form of each UbcH5B–Ub variant and measured its binding affinity for GSTRNF38-RING alone and with UbDGG using SPR. Alone, all three mutants were defective in binding GST-RNF38-RING compared with UbcH5BC85S–Ub or UbcH5B–Ub. UbDGG affected a similar trend as observed in the lysine discharge assays. Binding of UbcH5BC85S S108R–Ub for GST-RNF38-RING was not enhanced, whereas 5.6- and 4.6-fold increases in binding affinity were measured for UbcH5BC85S I88A–Ub and UbcH5BI37A–Ub, respectively, compared with 12-fold for UbcH5BC85S–Ub or UbcH5B–Ub (Table 1). Our data show that without E3, UbB effects are at best marginal; when UbcH5B or UbD variants are introduced that abrogate E3 function, UbB stimulation is also abolished, whereas variants that only impair E3 function are still stimulated by UbB, but to a lesser extent. Together our data suggest that the primary role of UbB is to support E3 function by stabilizing the E3-E2Ub complex. Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 5
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
UbB-Stimulated Ub Transfer Is RING Dependent Previously, ubiquitinated Mdm2 has been shown to facilitate Ub transfer in a backside-dependent manner—polyUb chains on the E3 are proposed to recruit UbcH5Ub via the backside interaction to catalyze direct Ub transfer to substrate in a RING-independent manner (Ranaweera and Yang, 2013). To probe the role of UbB in ubiquitination, we compared Ub transfer in pulse-chase assays using autoubiquitinated GST-RNF38-RING (Ubn–GSTRNF38-RING) and WT, UbcH5B S22R, UbcH5B F62A P95D, and UbcH5B S22R F62A P95D. The latter two mutants are defective in binding the RING domain and were used to investigate RING-independent Ub transfer. Pulsed UbcH5BUb variants were chased with Ubn–GST-RNF38-RING, and the simultaneous discharge of UbcH5BUb variants and transfer of Ub to Ubn–GST-RNF38-RING were followed (Figures S3A and S3B). Ub transfer was only observed for the RING-binding UbcH5B variants, and WT transferred Ub more rapidly than UbcH5B S22R (Figure 2A). Because GST is not a known biological substrate of RNF38-RING, these assays were repeated with Ubn– BIRC4-mediated Ub transfer to itself and to SMAC. As observed for Ubn–GST-RNF38-RING, Ub transfer only occurred with the RING-binding UbcH5B variants, and Ubn–BIRC4-mediated Ub transfer was faster with WT than UbcH5B S22R (Figures 2B, 2C, and S3C). There was no difference in Ub discharge to lysine by WT and UbcH5B S22R under identical pulsed-chased conditions unless K0UbDGG was present (Figure 1F), suggesting that polyUb chains on E3s and K0UbDGG perform the same role. Indeed, Ubn–BIRC4 discharged UbcH5BUb faster to lysine than unmodified BIRC4 (Figure S3D). Together, these data show that growing polyUb chains are sources for UbB but only in RING-dependent Ub transfer.
Figure 2. UbB Sources and Contributions to RING-Dependent and RING-Independent Ub Transfer (A) Non-reduced autoradiograms of pulsed-chased assays showing the simultaneous disappearance of UbcH5B32P-Ub variants and transfer of 32 P-Ub to Ubn–GST-RNF38-RING over time. The asterisk (*) is a mix of E1Ub and Ub-E1. (B) As in (A) but with Ubn–BIRC4. (C) Non-reduced autoradiograms showing the transfer of Ub to SMAC-32P over time by Ubn–BIRC4 in pulsed-chased reactions with indicated UbcH5BUb variants. (D) As in Figure 1F but using K0UbDGG or UbcH5BS22R F62A P95D–Ub and RNF38-RING.
6 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Stabilization of RNF38-RING-UbcH5BUb Directs UbB-Mediated Processivity UbB-mediated processivity is suggested to be dependent upon E2Ub oligomerization, where the Ub from one UbcH5Ub complex binds UbcH5’s backside in another UbcH5Ub complex to form a continuous chain (Brzovic et al., 2006; Sakata et al., 2010). To test whether UbcH5BUb is a source for UbB, we performed RNF38-RING-mediated lysine discharge assays of UbcH5BUb alone and with UbcH5BS22R F62A P95D–Ub, a stable isopeptide conjugate that can bind the backside of UbcH5BUb but not RNF38-RING or UbB. This UbcH5B–Ub variant promoted discharge of UbcH5BUb but was preferred over lysine as an acceptor (Figure 2D). Correspondingly, addition of UbcH5BS22R F62A P95D–Ub enhanced GST-RNF38-RING’s binding affinity for UbcH5B–Ub but not UbcH5BS22R–Ub (Table 1). These data show that UbcH5B–Ub enhances RNF38RING-UbcH5B–Ub binding and stimulates Ub transfer. Without E3, UbcH5CUb backside-associated oligomers have a KD 12-fold tighter than the noncovalent UbB-UbcH5C complex and are observed as higher molecular weight complexes at concentrations of 200 mM using gel filtration (E) Overlaid gel filtration Superdex 75 chromatograms of UbcH5B–Ub (red), UbcH5BS22R–Ub (blue), and gel filtration standards (GE Healthcare, orange and purple) with standard peaks and molecular weights indicated. See also Figure S3.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Figure 3. Ubn–BIRC4 and Ubn–SMAC Act as UbB Sources to Stimulate Ub Transfer (A) Non-reduced autoradiograms of pulsedchased assays showing the simultaneous disappearance of UbcH5B32P-K0Ub variants and transfer of 32P-K0Ub to BIRC4. The asterisk (*) is a mix of E1K0Ub and K0Ub-E1. (B) SDS-PAGE showing stopped BIRC4 and SMAC reactions used in (C). E1 was omitted from control reactions. (C) Non-reduced autoradiograms of pulsedchased lysine discharge assays showing disappearance of UbcH5B32P-Ub variants over time by E3/substrate mixtures shown in (B). (D) As in (C) but mediated by BIRC4 mixed with indicated SMAC variants.
absence of E3 suggest UbB is dependent on E3-E2Ub complex to mediate processivity.
chromatography (Brzovic et al., 2006). Although UbcH5B and UbcH5C vary in sequence by only four amino acids, to validate that UbcH5B behaves similarly, we performed analytical gel filtration chromatography on UbcH5B–Ub and UbcH5BS22R– Ub at concentrations of 200 mM. The elution volumes of both of these variants were approximately equivalent to carbonic anhydrase (29 kDa), suggesting both are monomers (Figure 2E). For UbcH5B–Ub, the largest higher molecular weight complex observed by gel filtration was a dimer and only when considerably higher concentrations and total quantities were loaded (Figure S3E). Our SPR analyses showed that GST-Ub bound UbcH5B–Ub with KD of 300 mM. Thus, although the UbBUbcH5B interaction (200 mM; Table 1) was stronger than the UbB-UbcH5B–Ub interaction, UbcH5B–Ub was still competent to bind UbB. These data show UbcH5B–Ub can function as a source of UbB to promote Ub transfer in vitro, but the high concentrations required to observe binding or dimerization in the
Ubiquitinated E3 and Substrate Mediate in cis UbB-Stimulated RING-Dependent Ub Transfer Based on these data, we propose that UbB-UbcH5B binding promotes processivity in an E3-dependent manner using free Ub, E2Ub, and/or Ub chains on the E3 as a source for UbB. To further investigate this mechanism, we performed pulse-chase Ub transfer assays with BIRC4, WT or UbcH5B S22R and K0Ub, which can multimonoubiquitinate a target but lacks lysines on which to build a polyUb chain. In early time points, the decrease in UbcH5BK0Ub variant and corresponding increase in BIRC4-K0Ubn were comparable, but in later time points, WT UbcH5BK0Ub discharged faster to BIRC4 (Figure 3A). To determine whether Ubn–BIRC4 acts as an in cis or in trans UbB source, we compared it with unmodified BIRC4 containing equivalent free Ub in lysine discharge assays. Ubn–BIRC4 discharged UbcH5BUb faster than BIRC4 and did not stimulate discharge of UbcH5B S22RUb (Figures 3B and 3C), suggesting that it acts as an in cis source of UbB. In vivo, substrate binding stabilizes some E3s by preventing autoubiquitination and subsequent degradation (Fredrickson et al., 2013). To investigate whether ubiquitinated substrate can also act as an UbB source to stimulate Ub transfer, we compared Ubn–SMAC/Ubn–BIRC4, Ubn–BIRC4, BIRC4/SMAC, and BIRC4 in lysine discharge assays. We generated BIRC4/ SMAC stocks by performing reactions with limited amounts of Ub (Figure 3B); no additional purification was carried out to enrich for select species. Ubn–SMAC/Ubn–BIRC4 and Ubn– BIRC4 both stimulated discharge of UbcH5BUb compared with BIRC4/SMAC and BIRC4 but not UbcH5B S22RUb Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 7
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
promote UbB-stimulated BIRC4-mediated discharge of UbcH5BUb and UbcH5B S22RUb to lysine. To differentiate between ubiquitinated SMAC acting as an in cis or in trans UbB source, we also tested SMAC variants containing the mutation A56M, which eliminates the SMAC-BIRC4 interaction (Chai et al., 2000). UbcH5BUb discharge was stimulated by SMAC–Ub1 and SMAC–Ub2 compared with SMAC and the SMAC A56M variants while UbcH5B S22RUb discharge was not stimulated by any SMAC variant (Figure 3D). These data suggest that ubiquitination is regulated in part by a positive feedback loop: once RING E3 or substrate is ubiquitinated, it can act as an in cis source of UbB, thereby upregulating Ub transfer by enhancing UbB-UbcH5BUb-RING E3 binding and increasing catalytic turnover.
Figure 4. Structures of E3-E2–Ub and E3-UbB-E2–Ub (A) Cartoon representation of the E3-E2–Ub complex. Left and right are related by 90 rotation about the y axis. UbcH5B is colored cyan, UbD yellow, and RNF38-RING gray. The UbcH5B–Ub linkage is indicated with an arrow and UbcH5B’s structural components labeled. (B) Cartoon representation of the E3-UbB-E2–Ub. UbB is colored orange and all other features are colored or highlighted as in (A). (C) Ribbon diagram showing superposition of E3-UbB-E2–Ub colored black with UbB omitted and both copies of E3-E2–Ub from the asymmetric unit colored red and wheat. The UbcH5B a1b1 loop is indicated by a black arrow. Left and right are related by 90 rotation about the y axis and the view is identical to those shown in (A) and (B). See also Figures S4 and S5.
(Figure 3C). Unmodified SMAC but not BIRC4 was present in the Ubn–SMAC/Ubn–BIRC4 reaction, so to exclude the possibility that only Ubn–BIRC4 contributed to UbB-stimulated discharge, we then tested the ability of SMAC with one or two Ub molecules at its C terminus (SMAC–Ub1 and SMAC–Ub2, respectively) to 8 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Mechanistic Insights into UbB-Mediated Stimulation Based on Structures of RNF38-UbcH5B–Ub Complex Alone and with UbB Current UbB-UbcH5 family complex structures reveal which protein-protein interactions mediate backside binding but lack insight into how these interactions stimulate Ub transfer. Given that UbD is essential for UbB-mediated stimulation, we reasoned that comparison of the crystal structures of a RING E3 bound to UbcH5B–Ub in the presence and absence of UbB might reveal the molecular mechanism(s) of stimulation. We crystallized complexes of RNF38-RING-UbcH5B–Ub alone and with UbB to 2.04 A˚ and 1.53 A˚, respectively (Figure 4; Table 2) using UbcH5BS22R–Ub to generate the UbB-free complex. There are two copies of RNF38-RING-UbcH5BS22R–Ub complex (designated E3-E2–Ub; rmsd of 0.332 A˚ for Ca atoms) and one copy of RNF38-RING-UbB-UbcH5B–Ub complex (designated E3UbB-E2–Ub) per asymmetric unit. In both structures, RNF38UbcH5B, RNF38-UbD, and UbD-UbcH5B interactions resemble those observed in other RING E3-E2–Ub complexes (Figures S4A–S4C); we mutated a selection of key residues in these interfaces and found they were all defective in our lysine discharge assays (Figures S4D–S4F). Two loops adjacent to both termini of the RING domain appear to grip UbD, thereby acting as the additional UbD stabilization component observed in other RING E3-E2–Ub complexes (Figures 4 and S4G–S4J). Thus, RNF38 uses an Ub transfer mechanism similar to other RING and U-box E3s. Overall, the structures and interactions of RNF38, UbcH5B, and UbD are similar when we superpose the structure of E3UbB-E2–Ub with both copies of E3-E2–Ub molecules in the asymmetric unit (root-mean-square deviation [rmsd] of 0.435 and 0.397 A˚ for Ca atoms, respectively; Figure 4C). The UbB-UbcH5B interactions resemble those observed in the NMR structure of UbB-UbcH5C complex (Brzovic et al., 2006) and in the crystal structures of UbcH5BC85S–Ub (Sakata et al., 2010) and UbB-UbcH5A (Bosanac et al., 2011) complexes (Figures S5A–S5D). UbB-UbcH5 interactions involve UbB’s Ile44 patch and UbcH5’s backside b1-3 surface surrounding Ser22 (Figures 5A and 5B). UbB’s Leu8 surface packs against UbcH5’s a1b1 and b2b3 loops (
Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin Graphical Abstract
Authors Lori Buetow, Mads Gabrielsen, ..., Brian O. Smith, Danny T. Huang
Correspondence [email protected]
In Brief Buetow et al. show that non-covalent ubiquitin binding to the backside of E2 stimulates RING E3-catalyzed initial ubiquitin transfer and poly-ubiquitin chain formation. Crystal structures and biochemical analyses reveal that backside bound ubiquitin stabilizes RING E3-E2ubiquitin complex for catalysis.
Highlights B
Accession Numbers D
d
Ub activates RING E3-UbcH5BUb complex for Ub transfer
d
Free Ub, E2Ub, and monoUb and polyUb chains on E3 and substrate act as UbB sources
d
d
UbB enhances RING E3-UbcH5B–Ub affinity, and RING E3UbcH5B–Ub improves UbB affinity Structure-based insights into UbB stimulatory mechanism
Buetow et al., 2015, Molecular Cell 58, 1–14 April 16, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.02.017
4V3K 4V3L
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Molecular Cell
Article Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin Lori Buetow,1 Mads Gabrielsen,1 Nahoum G. Anthony,2 Hao Dou,1,4 Amrita Patel,1 Hazel Aitkenhead,1 Gary J. Sibbet,1 Brian O. Smith,3 and Danny T. Huang1,* 1Cancer
Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK 3Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 4Present address: Institute of Medical Genetics, School of Medicine, Shandong University, No. 44 Wenhuaxi Road, Jinan, Shandong 250012, People’s Republic of China *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.02.017 2Strathclyde
SUMMARY
RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (UbB) enhances both RING-independent and RINGdependent UbcH5B-catalyzed donor Ub (UbD) transfer, but with a more prominent effect in RING-dependent transfer. UbB enhances RING E3s’ affinities for UbcH5B–Ub, and RING E3-UbcH5B–Ub complex improves UbB’s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B–Ub in the absence and presence of UbB, together with molecular dynamics simulation and biochemical analyses, suggests UbB restricts the flexibility of UbcH5B’s a1 and a1b1 loop. UbB supports E3 function by stabilizing the RING E3UbcH5B–Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, UbB serves as an allosteric activator of RING E3-mediated Ub transfer.
INTRODUCTION Post-translational modification by Ub plays a crucial role in regulating a plethora of events in eukaryotic cells by altering proteins’ half-lives, interacting partners, and activities. Ub modification is achieved by sequential actions of Ub-activating enzyme (E1), E2, and E3. E1 activates and transfers Ub’s C terminus to E2’s catalytic cysteine, forming an E2Ub thioester intermediate ( indicates a thioester). E3 recruits E2Ub and substrate to facilitate Ub transfer from E2 to a substrate lysine side chain or N-terminal methionine. There are four classes of E3s: RING, HECT, U-box, and RING-in-between-RING that utilize varying mechanisms to transfer Ub to substrate (Dye and Schulman, 2007; Spratt
et al., 2014). RING E3s comprise the largest family of E3s and function by recruiting E2Ub via the RING domain and substrate(s) via protein-protein interaction domain(s) to promote direct Ub transfer from E2 to substrate. RING E3s lack active sites, instead functioning as scaffolds. Biochemical and recent structural studies have shown that RING domains bind E2Ub, restraining UbD in a closed configuration where numerous RING-UbD and E2-UbD interactions optimally position UbD’s C-terminal tail within E2’s active site cleft and orient the thioester for nucleophilic attack (Dou et al., 2012; Plechanovova´ et al., 2012; Pruneda et al., 2012; Saha et al., 2011; Wickliffe et al., 2011). Although the RING domain is sufficient to populate E2Ub in the closed active configuration, an additional component outside the RING domain helps maintain this state and enhances the catalytic efficiency of Ub transfer (Dou et al., 2013; Plechanovova´ et al., 2012). Examples include the phosphorylated linker tyrosine in Cbl (Dou et al., 2013) and the C-terminal tail of RING E3 dimers (Dou et al., 2012; Plechanovova´ et al., 2012). Residues aligning the E2 active site and the chemical environment of the acceptor amine also influence RING E3-catalyzed Ub transfer (Berndsen et al., 2013; Ozkan et al., 2005; Scott et al., 2014; Wickliffe et al., 2011; Wu et al., 2003; Yunus and Lima, 2006). Furthermore, some RING E3s such as gp78, Cue1p, and Rad18 harbor additional E2-binding motifs that bind the backside of Ube2g2, Ubc7p, and Rad6, respectively (Das et al., 2009, 2013; Hibbert et al., 2011; Li et al., 2009; Metzger et al., 2013). In gp78 and Cue1p, the additional E2-binding element modulates RING E3-E2 binding affinity to facilitate Ub transfer, whereas the additional E2-binding motif in Rad18 serves to direct monoubiquitination. Along with E2-binding motifs on select E3s, Ub has also been shown to bind to the backside of several E2s, including Rad6, UbcH5, UbcH6, and Ube2g2 families (Bocik et al., 2011; Brzovic et al., 2006; Hibbert et al., 2011; Miura et al., 1999). This backside interaction is important for processivity in polyUb chain formation and Ub transfer to substrate (Brzovic et al., 2006; Ranaweera and Yang, 2013). For the UbcH5 family, UbB is proposed to facilitate processivity by forming self-assembled oligomers of UbcH5Ub, thereby increasing local concentrations of substrate and/or by bridging the gap between the E2- and substrate-binding domains of the E3 (Brzovic et al., 2006; Page Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 1
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Figure 1. UbB Stimulates RING-Dependent and RING-Independent Ub Transfer (A) Reduced autoradiogram showing autoubiquitination by GST-RNF38-RING with 32P-Ub and UbcH5B variants over time. The asterisk (*) represents nonreducible Ub-E1. (B) As in (A) but for full-length BIRC4 instead of GST-RNF38-RING. (C) Reduced autoradiogram showing ubiquitination of SMAC-32P by BIRC4 and UbcH5B variants over time. (D) Reduced autoradiograms showing diUb formation over time by UbcH5B variants with (top) or without RNF38-RING (bottom). Different exposure times were used for the panels. (E) Kinetics of diUb formation by RNF38-RING with varying concentrations of WT and UbcH5B S22R. Three rate replicates were measured for each UbcH5B concentration. Kinetic parameters and 95% CI are indicated. Error bars represent SD. (legend continued on next page)
2 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
et al., 2012; Sakata et al., 2010). In addition, polyUb chains on E3s purportedly serve as scaffolds for backside binding UbcH5Ub to mediate RING-independent Ub transfer to substrate (Ranaweera and Yang, 2013). Which of these mechanisms predominates, if any, is unknown and requires further investigation. Structures of UbcH5 bound non-covalently to Ub reveal that Ub’s Ile44 surface interacts with UbcH5’s b1-3 surface centering on Ser22 (Brzovic et al., 2006). Disruption of either surface hinders RING E3-catalyzed polyUb chain formation but the effect on backside-mediated Ub transfer has not been thoroughly characterized. Here, we uncover an original role for UbB in promoting ubiquitination. We show that UbB directly simulates RING-independent and RING-dependent Ub transfer. Using the RING domain of RNF38, an E3 ligase implicated in p53 ubiquitination (Sheren and Kassenbrock, 2013), as a model system, we determine crystal structures of RNF38-RING bound to UbcH5B–Ub alone and in complex with free Ub bound to UbcH5B’s backside. Comparison of the structures, together with other biophysical and biochemical analyses, reveals that UbB alters UbcH5B dynamics and transforms RNF38-UbcH5B–Ub into a higher affinity complex with improved catalytic efficiency. Our results demonstrate UbB functions as an allosteric activator of RING E3-E2Ub complexes to enhance Ub transfer. RESULTS UbB-UbcH5B Interaction Stimulates RING-Independent and RING-Dependent Ub Transfer For the UbcH5 family of E2s, S22R substitution abrogates UbB interactions and is defective in RING E3-catalyzed polyUb chain formation (Brzovic et al., 2006). To validate these findings, we performed ubiquitination assays with wild-type UbcH5B (WT) and UbcH5B S22R on N-terminally glutathione-S-transferase tagged RNF38 RING domain (GST-RNF38-RING), full-length BIRC4 and the BIRC4 substrate SMAC. Polyubiquitination or multimonoubiquitination of GST-RNF38-RING, BIRC4, and SMAC were diminished with UbcH5B S22R compared with WT (Figures 1A–1C) but not essentially eliminated as observed in the BRCA1 autoubiquitination assays (Brzovic et al., 2006). During Ub transfer, RING E3 binds E2Ub, optimizes UbD in the closed active configuration, and positions substrate or acceptor Ub (UbA) for catalysis. To assess the effects of UbB on Ub transfer, we performed in vitro single turnover diUb formation assays using WT and UbcH5B S22R; with surface plasmon resonance (SPR), we estimated a KD of 200 mM for the UbBUbcH5B interaction (Figure S1; Table 1), similar to the reported KD for the UbB-UbcH5C interaction (300 mM; Brzovic et al.,
2006). UbcH5B variants were precharged with equimolar concentrations of 32P-Ub and then chased by adding saturated Ub lacking the C-terminal Gly-Gly motif (UbDGG), which cannot be charged by Ub E1. Surprisingly, UbcH5B S22R was slower in diUb formation whether E3 was present or not (Figure 1D), but the overall effects were more obvious with E3 (Figure S2A). We then measured the kinetic constants of diUb formation with RNF38-RING and found Km decreased 8-fold, while kcat increased 2-fold for WT compared with UbcH5B S22R (Figures 1E and S2B). To exclude potential UbB mediated effects on stabilizing or recruiting UbA within the E-S intermediate complex, we tested single-turnover Ub transfer to lysine in pulse chase assays with and without 300 mM K0UbDGG, where all seven lysines were mutated to arginine to prevent Ub acting as an acceptor. In the absence of K0UbDGG, regardless of whether RNF38-RING was present, the rates of UbcH5BUb and UbcH5B S22RUb discharge were comparable, whereas including K0UbDGG increased the rate of discharge for UbcH5BUb but not for UbcH5B S22RUb (Figure 1F). Because of the quick rate of discharge and large variability, quantification of stimulation was not possible. Although lysine concentrations differed between assays with and without RNF38-RING (10 mM and 150 mM, respectively), effects were more obvious when E3 was present. To test the specificity of the UbB-UbcH5B interaction, we then performed our lysine discharge assays with K0UbDGG I44A since this mutation also abrogates the UbBUbcH5B interaction. K0UbDGG I44A did not promote UbcH5BUb discharge under any condition tested, and the rate of discharge was similar to assays performed with UbcH5B S22RUb, suggesting that both UbcH5B S22R and K0UbDGG I44A exclusively impact backside-mediated stimulation (Figure 1F). Furthermore, we also tested a dimeric RING E3, BIRC4, and a U-box E3, UBE4B, and found that K0UbDGG promoted UbcH5BUb discharge in both E3-mediated reactions (Figure 1G), establishing that this feature is not exclusive to RNF38-RING. Together, these data demonstrate that UbB plays a distinct role in stimulating Ub transfer unrelated to acting as a donor or acceptor. This effect occurs in a RING-dependent and RING-independent manner, but is more pronounced in the presence of a RING domain. UbB functions to stabilize the E-S intermediate complex when RNF38-RING is present, suggesting that the binding affinity between the RING domain and UbcH5BUb might be enhanced. UbB-RING E3 Synergy Enhances UbcH5B–Ub Binding Interactions To investigate whether UbB improves the binding affinity of RNF38-RING for UbcH5BUb, we performed SPR analyses to
(F) Non-reduced autoradiograms of pulsed-chased reactions showing the disappearance of UbcH5B32P-Ub variants over time with lysine in the presence and absence of K0UbDGG or K0UbDGG I44A, as indicated with RNF38-RING (top) or without E3 (bottom). (G) As for (F) but with UbcH5B32P-Ub, K0UbDGG, the RING domains of RNF38 (top) and BIRC4 (middle) and UBE4B U-box (bottom). (H) Non-reduced antibiotin immunoblots of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5BUbC over time with K0UbDGG and RNF38-RING as indicated. (I) Non-reduced autoradiograms of pulsed-chased lysine discharge reactions showing the disappearance of UbcH5B32P-Ub variants over time with (left) or without RNF38-RING (right) in the presence and absence of K0UbDGG. See also Figure S2.
Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 3
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Table 1. KD for Interactions between RING E3 Variants, UbcH5B Variants, UbcH5B–Ub Variants, and Ub Immobilized Protein
Analytea
KD (mM)
Binding Enhancement (Fold)b
GST-RING E3 RNF38-RING
UbcH5B
89 ± 1
RNF38-RING
UbcH5B + 600 mM UbDGG
73 ± 2
RNF38-RING
UbcH5B–Ub
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BS22R–Ub
RNF38-RING
UbcH5BS22R–Ub + 600 mM UbDGG
1.3 ± 0.1
RNF38-RING
UbcH5B–Ub + 3 mM UbDGG
0.5 ± 0.01
RNF38-RING
UbcH5B–Ub + 10 mM UbDGG
0.33 ± 0.01
RNF38-RING
UbcH5B–Ub + 30 mM UbDGG
0.22 ± 0.01
RNF38-RING
UbcH5B–Ub + 100 mM UbDGG
0.11 ± 0.01
RNF38-RING
UbcH5BE9D–Ub
0.37 ± 0.04
RNF38-RING
UbcH5BE9D–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BQ20A–Ub
RNF38-RING
UbcH5BQ20A–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BI37A–Ub
RNF38-RING
UbcH5BI37A–Ub + 600 mM UbDGG
0.79 ± 0.05
4.6
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG L8A
0.13 ± 0.02
7.1
RNF38-RING
UbcH5B–Ub + 100 mM UbcH5BS22R,F62A,P95D–Ub
0.20 ± 0.01
RNF38-RING
UbcH5BS22R–Ub + 100 mM UbcH5BS22R,F62A,P95D–Ub
0.94 ± 0.02
1.2
0.92 ± 0.08
C85S
–Ub
0.077 ± 0.006
11.9
1.04 ± 0.12
0.046 ± 0.003
0.8
8.0
2.2 ± 0.4 0.26 ± 0.02
8.5
3.6 ± 0.6
4.6 ± 0.2
RNF38-RING
UbcH5B
RNF38-RING
UbcH5BC85S–Ub + 600 mM UbDGG
RNF38-RING
UbcH5BC85S,I88A–Ub
27 ± 1
RNF38-RING
UbcH5BC85S,I88A–Ub + 600 mM UbDGG
4.8 ± 0.1
RNF38-RING
UbcH5BC85S,S108R–Ub
74 ± 2
RNF38-RING
UbcH5BC85S,S108R–Ub + 600 mM UbDGG
BIRC4-RING
UbcH5B–Ub
BIRC4-RING
UbcH5B–Ub + 600 mM UbDGG
BIRC4-RING
UbcH5BS22R–Ub
33.8 ± 2.4
BIRC4-RING
UbcH5BS22R–Ub + 600 mM UbDGG
36.5 ± 2.4
UBE4B-U-box
UbcH5B–Ub
29 ± 0.5
UBE4B-U-box
UbcH5B–Ub + 600 mM UbDGG
4.9 ± 0.3
0.36 ± 0.01
76 ± 2
12.8 5.6 1.0
39.1 ± 1.3
S22R
6.2 ± 0.6
UBE4B-U-box
UbcH5B
UBE4B-U-box
UbcH5BS22R–Ub + 600 mM UbDGG
44.8 ± 1.4
Ub
UbcH5B
206 ± 6
Ub
UbcH5B–Ub
284 ± 8
Ub
UbcH5B–Ub + molar excess of RNF38-RING
Ub
RNF38-RING
Ub
UbcH5B Q20A
197 ± 17
Ub L8A
UbcH5B
296 ± 7
Ub L8A
UbcH5B–Ub
353 ± 7
Ub L8A
UbcH5B–Ub + molar excess of RNF38-RING
–Ub
6.3 0.92 5.9
44.2 ± 1.7 0.99
GST-Ub
14 ± 1.8
20
no binding
44 ± 3
8
MBP-tag RNF38-RING
UbcH5B–Ub
2.04 ± 0.05
RNF38-RING
UbcH5B–Ub + 600 mM UbDGG
0.08 ± 0.007
26 (Continued on next page)
4 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Table 1.
Continued
Immobilized Protein
Analytea
Ub
UbcH5B–Ub
137 ± 11
Ub
UbcH5B–Ub + molar excess of RNF38-RING
4.9 ± 1.4
KD (mM)
Binding Enhancement (Fold)b 28
SEM is indicated. Number of replicates, representative sensorgrams, and binding curves are shown in Figure S1. a Analytes containing fixed concentration of UbDGG variants, UbcH5BS22R,F62A,P95D–Ub or RNF38-RING are indicated. b The degree of binding enhancement in the presence of UbDGG variant or RNF38-RING was determined by using the KD in the absence of UbDGG variant or RNF38-RING divided by the KD in the presence of UbDGG variant or RNF38-RING, respectively.
measure the binding affinity of GST-RNF38-RING for stably linked UbcH5B–Ub alone and with 600 mM UbDGG. We generated stable conjugates by mutating UbcH5B’s catalytic cysteine to a lysine or serine, thereby mimicking UbcH5BUb by forming, respectively, a stable isopeptide (UbcH5B–Ub) or oxyester (UbcH5BC85S–Ub) linkage with Ub. Though UbcH5B– Ub binds slightly tighter than UbcH5BC85S–Ub, in both instances, UbDGG improved binding affinity by 12-fold (Table 1). We then measured GST-RNF38-RING’s binding affinity for UbcH5BS22R– Ub and found no improvement upon including UbDGG (Table 1). To ensure that GST dimerization did not contribute to binding enhancement, we determined the binding affinity of MBPRNF38-RING for UbcH5B–Ub under similar conditions by SPR and found UbDGG enhanced binding by 26-fold (Table 1). When we performed the same experiments with GST-BIRC4RING and GST-UBE4B-U-box, UbDGG improved binding affinities for UbcH5B–Ub by 6-fold but not for UbcH5BS22R–Ub (Table 1). Thus UbB enhances RING/U-box E3-UbcH5B–Ub interactions but the extent of enhancement depends on the complex. Interestingly, when the same experiments were performed using UbcH5B lacking conjugated Ub, addition of UbDGG only improved RNF38-RING’s binding affinity for UbcH5B by 1.2fold (Table 1), suggesting that the presence of UbD is required for UbB-mediated binding enhancement. Since UbB improved RNF38’s binding affinity for UbcH5B–Ub, we asked whether RNF38 affects UbB-UbcH5B–Ub binding affinity. We performed SPR analyses to determine GST-RNF38RING’s binding affinity for UbcH5B–Ub with varying UbDGG concentrations. Addition of 3 mM UbDGG marginally increased RNF38-RING’s binding affinity for UbcH5B–Ub, and this binding affinity progressively improved with increasing UbDGG concentrations, approaching saturation at 100 mM UbDGG (Table 1). Consistent with the SPR results, the rate of RNF38-mediated UbcH5BUb lysine discharge with increasing K0UbDGG concentrations approached saturation near 100 mM K0UbDGG (Figure S2C). To determine the KD of UbB for RNF38-UbcH5B–Ub, we used SPR to measure GST-Ub’s binding affinity for UbcH5B–Ub alone and with saturated RNF38-RING. Without RNF38-RING, GST-Ub displayed weak binding affinities for UbcH5B–Ub (284 mM) and UbcH5B (206 mM); remarkably, RNF38-RING enhanced GST-Ub’s binding affinity for UbcH5B–Ub by 20fold; similarly, the addition of UbDGG enhanced the binding affinity of MBP-Ub for UbcH5B–Ub by 28-fold (Table 1). Activation of RING E3-UbcH5BUb by UbB RING E3s lock E2Ub into a closed conformation (Dou et al., 2012; Plechanovova´ et al., 2012; Pruneda et al., 2012) and
induce global changes in E2 (Benirschke et al., 2010) to activate the E2Ub thioester bond for catalysis. We questioned how UbB influences this mechanism. The globular body of UbD contributes the bulk of UbD interactions with UbcH5B and RING E3 and is required for E3-induced active site conformational changes, so we initially investigated whether the globular body of UbD is also important for UbB stimulation. We performed lysine discharge assays on UbcH5B charged with a biotin-labeled Ub C-terminal peptide (residues 71–76; UbC) lacking the globular Ub body. Discharge of UbcH5BUbC was not mediated by RNF38-RING or stimulated by K0UbDGG (Figure 1H). Thus, the globular body of UbD is required for RING E3-mediated catalysis and UbB-stimulated Ub transfer. Next we examined UbcH5B mutants with well-characterized defects in E3-mediated Ub transfer. We used UbcH5B I88A (an a2 residue that contacts UbD’s tail), S108R (an a3 residue that contacts UbD’s Ile44 surface), and I37A (Ile37 is on the b sheet abutting the UbcH5B a3-UbD Ile44 interaction surface) substitutions in our lysine discharge assays. As a control, we included UbcH5B E140A; Glu140 is a surface residue and does not contact any interacting partners in available E3-E2–Ub complexes. UbcH5B E140A behaved comparably to WT UbcH5B in all conditions. K0UbDGG did not stimulate discharge of any mutant in the absence of E3. With RNF38-RING, although Ub discharge was impaired by UbcH5B I88A and I37A, addition of K0UbDGG still had a stimulatory effect, whereas UbcH5B S108R abolished both E3-mediation and K0UbDGG-stimulation of Ub transfer (Figures 1I and S2D). Whether UbcH5B I37A and I88A simply impaired E3-facilitated discharge or also caused defects in UbB-mediated stimulation was unclear, so we generated a stably linked form of each UbcH5B–Ub variant and measured its binding affinity for GSTRNF38-RING alone and with UbDGG using SPR. Alone, all three mutants were defective in binding GST-RNF38-RING compared with UbcH5BC85S–Ub or UbcH5B–Ub. UbDGG affected a similar trend as observed in the lysine discharge assays. Binding of UbcH5BC85S S108R–Ub for GST-RNF38-RING was not enhanced, whereas 5.6- and 4.6-fold increases in binding affinity were measured for UbcH5BC85S I88A–Ub and UbcH5BI37A–Ub, respectively, compared with 12-fold for UbcH5BC85S–Ub or UbcH5B–Ub (Table 1). Our data show that without E3, UbB effects are at best marginal; when UbcH5B or UbD variants are introduced that abrogate E3 function, UbB stimulation is also abolished, whereas variants that only impair E3 function are still stimulated by UbB, but to a lesser extent. Together our data suggest that the primary role of UbB is to support E3 function by stabilizing the E3-E2Ub complex. Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 5
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
UbB-Stimulated Ub Transfer Is RING Dependent Previously, ubiquitinated Mdm2 has been shown to facilitate Ub transfer in a backside-dependent manner—polyUb chains on the E3 are proposed to recruit UbcH5Ub via the backside interaction to catalyze direct Ub transfer to substrate in a RING-independent manner (Ranaweera and Yang, 2013). To probe the role of UbB in ubiquitination, we compared Ub transfer in pulse-chase assays using autoubiquitinated GST-RNF38-RING (Ubn–GSTRNF38-RING) and WT, UbcH5B S22R, UbcH5B F62A P95D, and UbcH5B S22R F62A P95D. The latter two mutants are defective in binding the RING domain and were used to investigate RING-independent Ub transfer. Pulsed UbcH5BUb variants were chased with Ubn–GST-RNF38-RING, and the simultaneous discharge of UbcH5BUb variants and transfer of Ub to Ubn–GST-RNF38-RING were followed (Figures S3A and S3B). Ub transfer was only observed for the RING-binding UbcH5B variants, and WT transferred Ub more rapidly than UbcH5B S22R (Figure 2A). Because GST is not a known biological substrate of RNF38-RING, these assays were repeated with Ubn– BIRC4-mediated Ub transfer to itself and to SMAC. As observed for Ubn–GST-RNF38-RING, Ub transfer only occurred with the RING-binding UbcH5B variants, and Ubn–BIRC4-mediated Ub transfer was faster with WT than UbcH5B S22R (Figures 2B, 2C, and S3C). There was no difference in Ub discharge to lysine by WT and UbcH5B S22R under identical pulsed-chased conditions unless K0UbDGG was present (Figure 1F), suggesting that polyUb chains on E3s and K0UbDGG perform the same role. Indeed, Ubn–BIRC4 discharged UbcH5BUb faster to lysine than unmodified BIRC4 (Figure S3D). Together, these data show that growing polyUb chains are sources for UbB but only in RING-dependent Ub transfer.
Figure 2. UbB Sources and Contributions to RING-Dependent and RING-Independent Ub Transfer (A) Non-reduced autoradiograms of pulsed-chased assays showing the simultaneous disappearance of UbcH5B32P-Ub variants and transfer of 32 P-Ub to Ubn–GST-RNF38-RING over time. The asterisk (*) is a mix of E1Ub and Ub-E1. (B) As in (A) but with Ubn–BIRC4. (C) Non-reduced autoradiograms showing the transfer of Ub to SMAC-32P over time by Ubn–BIRC4 in pulsed-chased reactions with indicated UbcH5BUb variants. (D) As in Figure 1F but using K0UbDGG or UbcH5BS22R F62A P95D–Ub and RNF38-RING.
6 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Stabilization of RNF38-RING-UbcH5BUb Directs UbB-Mediated Processivity UbB-mediated processivity is suggested to be dependent upon E2Ub oligomerization, where the Ub from one UbcH5Ub complex binds UbcH5’s backside in another UbcH5Ub complex to form a continuous chain (Brzovic et al., 2006; Sakata et al., 2010). To test whether UbcH5BUb is a source for UbB, we performed RNF38-RING-mediated lysine discharge assays of UbcH5BUb alone and with UbcH5BS22R F62A P95D–Ub, a stable isopeptide conjugate that can bind the backside of UbcH5BUb but not RNF38-RING or UbB. This UbcH5B–Ub variant promoted discharge of UbcH5BUb but was preferred over lysine as an acceptor (Figure 2D). Correspondingly, addition of UbcH5BS22R F62A P95D–Ub enhanced GST-RNF38-RING’s binding affinity for UbcH5B–Ub but not UbcH5BS22R–Ub (Table 1). These data show that UbcH5B–Ub enhances RNF38RING-UbcH5B–Ub binding and stimulates Ub transfer. Without E3, UbcH5CUb backside-associated oligomers have a KD 12-fold tighter than the noncovalent UbB-UbcH5C complex and are observed as higher molecular weight complexes at concentrations of 200 mM using gel filtration (E) Overlaid gel filtration Superdex 75 chromatograms of UbcH5B–Ub (red), UbcH5BS22R–Ub (blue), and gel filtration standards (GE Healthcare, orange and purple) with standard peaks and molecular weights indicated. See also Figure S3.
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
Figure 3. Ubn–BIRC4 and Ubn–SMAC Act as UbB Sources to Stimulate Ub Transfer (A) Non-reduced autoradiograms of pulsedchased assays showing the simultaneous disappearance of UbcH5B32P-K0Ub variants and transfer of 32P-K0Ub to BIRC4. The asterisk (*) is a mix of E1K0Ub and K0Ub-E1. (B) SDS-PAGE showing stopped BIRC4 and SMAC reactions used in (C). E1 was omitted from control reactions. (C) Non-reduced autoradiograms of pulsedchased lysine discharge assays showing disappearance of UbcH5B32P-Ub variants over time by E3/substrate mixtures shown in (B). (D) As in (C) but mediated by BIRC4 mixed with indicated SMAC variants.
absence of E3 suggest UbB is dependent on E3-E2Ub complex to mediate processivity.
chromatography (Brzovic et al., 2006). Although UbcH5B and UbcH5C vary in sequence by only four amino acids, to validate that UbcH5B behaves similarly, we performed analytical gel filtration chromatography on UbcH5B–Ub and UbcH5BS22R– Ub at concentrations of 200 mM. The elution volumes of both of these variants were approximately equivalent to carbonic anhydrase (29 kDa), suggesting both are monomers (Figure 2E). For UbcH5B–Ub, the largest higher molecular weight complex observed by gel filtration was a dimer and only when considerably higher concentrations and total quantities were loaded (Figure S3E). Our SPR analyses showed that GST-Ub bound UbcH5B–Ub with KD of 300 mM. Thus, although the UbBUbcH5B interaction (200 mM; Table 1) was stronger than the UbB-UbcH5B–Ub interaction, UbcH5B–Ub was still competent to bind UbB. These data show UbcH5B–Ub can function as a source of UbB to promote Ub transfer in vitro, but the high concentrations required to observe binding or dimerization in the
Ubiquitinated E3 and Substrate Mediate in cis UbB-Stimulated RING-Dependent Ub Transfer Based on these data, we propose that UbB-UbcH5B binding promotes processivity in an E3-dependent manner using free Ub, E2Ub, and/or Ub chains on the E3 as a source for UbB. To further investigate this mechanism, we performed pulse-chase Ub transfer assays with BIRC4, WT or UbcH5B S22R and K0Ub, which can multimonoubiquitinate a target but lacks lysines on which to build a polyUb chain. In early time points, the decrease in UbcH5BK0Ub variant and corresponding increase in BIRC4-K0Ubn were comparable, but in later time points, WT UbcH5BK0Ub discharged faster to BIRC4 (Figure 3A). To determine whether Ubn–BIRC4 acts as an in cis or in trans UbB source, we compared it with unmodified BIRC4 containing equivalent free Ub in lysine discharge assays. Ubn–BIRC4 discharged UbcH5BUb faster than BIRC4 and did not stimulate discharge of UbcH5B S22RUb (Figures 3B and 3C), suggesting that it acts as an in cis source of UbB. In vivo, substrate binding stabilizes some E3s by preventing autoubiquitination and subsequent degradation (Fredrickson et al., 2013). To investigate whether ubiquitinated substrate can also act as an UbB source to stimulate Ub transfer, we compared Ubn–SMAC/Ubn–BIRC4, Ubn–BIRC4, BIRC4/SMAC, and BIRC4 in lysine discharge assays. We generated BIRC4/ SMAC stocks by performing reactions with limited amounts of Ub (Figure 3B); no additional purification was carried out to enrich for select species. Ubn–SMAC/Ubn–BIRC4 and Ubn– BIRC4 both stimulated discharge of UbcH5BUb compared with BIRC4/SMAC and BIRC4 but not UbcH5B S22RUb Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc. 7
Please cite this article in press as: Buetow et al., Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.02.017
promote UbB-stimulated BIRC4-mediated discharge of UbcH5BUb and UbcH5B S22RUb to lysine. To differentiate between ubiquitinated SMAC acting as an in cis or in trans UbB source, we also tested SMAC variants containing the mutation A56M, which eliminates the SMAC-BIRC4 interaction (Chai et al., 2000). UbcH5BUb discharge was stimulated by SMAC–Ub1 and SMAC–Ub2 compared with SMAC and the SMAC A56M variants while UbcH5B S22RUb discharge was not stimulated by any SMAC variant (Figure 3D). These data suggest that ubiquitination is regulated in part by a positive feedback loop: once RING E3 or substrate is ubiquitinated, it can act as an in cis source of UbB, thereby upregulating Ub transfer by enhancing UbB-UbcH5BUb-RING E3 binding and increasing catalytic turnover.
Figure 4. Structures of E3-E2–Ub and E3-UbB-E2–Ub (A) Cartoon representation of the E3-E2–Ub complex. Left and right are related by 90 rotation about the y axis. UbcH5B is colored cyan, UbD yellow, and RNF38-RING gray. The UbcH5B–Ub linkage is indicated with an arrow and UbcH5B’s structural components labeled. (B) Cartoon representation of the E3-UbB-E2–Ub. UbB is colored orange and all other features are colored or highlighted as in (A). (C) Ribbon diagram showing superposition of E3-UbB-E2–Ub colored black with UbB omitted and both copies of E3-E2–Ub from the asymmetric unit colored red and wheat. The UbcH5B a1b1 loop is indicated by a black arrow. Left and right are related by 90 rotation about the y axis and the view is identical to those shown in (A) and (B). See also Figures S4 and S5.
(Figure 3C). Unmodified SMAC but not BIRC4 was present in the Ubn–SMAC/Ubn–BIRC4 reaction, so to exclude the possibility that only Ubn–BIRC4 contributed to UbB-stimulated discharge, we then tested the ability of SMAC with one or two Ub molecules at its C terminus (SMAC–Ub1 and SMAC–Ub2, respectively) to 8 Molecular Cell 58, 1–14, April 16, 2015 ª2015 Elsevier Inc.
Mechanistic Insights into UbB-Mediated Stimulation Based on Structures of RNF38-UbcH5B–Ub Complex Alone and with UbB Current UbB-UbcH5 family complex structures reveal which protein-protein interactions mediate backside binding but lack insight into how these interactions stimulate Ub transfer. Given that UbD is essential for UbB-mediated stimulation, we reasoned that comparison of the crystal structures of a RING E3 bound to UbcH5B–Ub in the presence and absence of UbB might reveal the molecular mechanism(s) of stimulation. We crystallized complexes of RNF38-RING-UbcH5B–Ub alone and with UbB to 2.04 A˚ and 1.53 A˚, respectively (Figure 4; Table 2) using UbcH5BS22R–Ub to generate the UbB-free complex. There are two copies of RNF38-RING-UbcH5BS22R–Ub complex (designated E3-E2–Ub; rmsd of 0.332 A˚ for Ca atoms) and one copy of RNF38-RING-UbB-UbcH5B–Ub complex (designated E3UbB-E2–Ub) per asymmetric unit. In both structures, RNF38UbcH5B, RNF38-UbD, and UbD-UbcH5B interactions resemble those observed in other RING E3-E2–Ub complexes (Figures S4A–S4C); we mutated a selection of key residues in these interfaces and found they were all defective in our lysine discharge assays (Figures S4D–S4F). Two loops adjacent to both termini of the RING domain appear to grip UbD, thereby acting as the additional UbD stabilization component observed in other RING E3-E2–Ub complexes (Figures 4 and S4G–S4J). Thus, RNF38 uses an Ub transfer mechanism similar to other RING and U-box E3s. Overall, the structures and interactions of RNF38, UbcH5B, and UbD are similar when we superpose the structure of E3UbB-E2–Ub with both copies of E3-E2–Ub molecules in the asymmetric unit (root-mean-square deviation [rmsd] of 0.435 and 0.397 A˚ for Ca atoms, respectively; Figure 4C). The UbB-UbcH5B interactions resemble those observed in the NMR structure of UbB-UbcH5C complex (Brzovic et al., 2006) and in the crystal structures of UbcH5BC85S–Ub (Sakata et al., 2010) and UbB-UbcH5A (Bosanac et al., 2011) complexes (Figures S5A–S5D). UbB-UbcH5 interactions involve UbB’s Ile44 patch and UbcH5’s backside b1-3 surface surrounding Ser22 (Figures 5A and 5B). UbB’s Leu8 surface packs against UbcH5’s a1b1 and b2b3 loops (
