Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of Gp78

Molecular Cell Article

Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Deﬁned Speciﬁc E2-Binding Region of gp78

Ranabir Das,1 Jennifer Mariano,2 Yien Che Tsai,2,4 Ravi C. Kalathur,3,4 Zlatka Kostova,2 Jess Li,1 Sergey G. Tarasov,1 Robert L. McFeeters,1 Amanda S. Altieri,1 Xinhua Ji,3 R. Andrew Byrd,1,* and Allan M. Weissman2,* 1Structural Biophysics Laboratory 2Laboratory of Protein Dynamics and Signaling 3Macromolecular Crystallography Laboratory Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA 4These authors contributed equally to this work *Correspondence: [email protected] (R.A.B.), [email protected] (A.M.W.) DOI 10.1016/j.molcel.2009.05.010

SUMMARY characterized by a conserved 350 amino acid catalytic domain. HECT E3s are catalytic intermediates in substrate ubiquitylation The activity of RING finger ubiquitin ligases (E3) is as a consequence of the transthiolation of ubiquitin from bound dependent on their ability to facilitate transfer of E2 to their conserved catalytic Cys (Fang and Weissman, 2004). ubiquitin from ubiquitin-conjugating enzymes (E2) RING finger and RING finger-like E3s collectively represent the to substrates. The G2BR domain within the E3 gp78 large majority of E3s. The RING finger is a compact Zn-binding binds selectively and with high affinity to the E2 domain of 40 to 100 amino acids. These domains generally Ube2g2. Through structural and functional analyses, bind E2s with low affinity and do not form catalytic intermediates with ubiquitin. It is generally believed that RING fingers either we determine that this occurs on a region of Ube2g2 position E2Ub to facilitate transfer to substrates or function distinct from binding sites for ubiquitin-activating as allosteric activators of E2Ub (Lorick et al., 2005; Ozkan enzyme (E1) and RING fingers. Binding to the G2BR et al., 2005). Binding sites on E2s for RING fingers, HECT results in conformational changes in Ube2g2 that domains, and E1 all overlap. Therefore, E2s must dissociate affect ubiquitin loading. The Ube2g2:G2BR interac- from ligase domains to reload with ubiquitin (Huang et al., tion also causes an 50-fold increase in affinity 2005; Eletr et al., 2005). Interestingly, there are a few examples between the E2 and RING finger. This results in mark- in which other regions, either within a multisubunit ubiquitin edly increased ubiquitylation by Ube2g2 and the ligase complex or a single subunit E3, bind specific E2s through gp78 RING finger. The significance of this G2BR generally uncharacterized interactions (Madura et al., 1993; Ha- effect is underscored by enhanced ubiquitylation takeyama et al., 1997; Wu et al., 2002; Biederer et al., 1997; Chen observed when Ube2g2 is paired with other RING et al., 2006). This could increase the availability of E2Ub and theoretically allow for reloading of E2 with ubiquitin without finger E3s. These findings uncover a mechanism dissociation from the E3. whereby allosteric effects on an E2 enhance E2- Ubiquitylation and proteasomal degradation perform critical RING finger interactions and, consequently, ubiqui- functions in degradation of misfolded, unassembled, and highly tylation. regulated proteins from the endoplasmic reticulum (ER). ER- associated degradation (ERAD) is a multistep, highly coordi- INTRODUCTION nated process (Nakatsukasa and Brodsky, 2008). In mammals, there are at least five known ER membrane-spanning ERAD Ubiquitylation occurs as the result of a hierarchical multienzyme E3s. Among these is gp78, also known as the human tumor au- process. Ubiquitin-activating enzyme (E1) activates ubiquitin, tocrine motility factor receptor (AMFR). gp78 is implicated in forming a thiolester linkage between the active site Cys of E1 degradation of T cell antigen receptor subunits, regulatory and the C terminus of ubiquitin. Ubiquitin is transferred to the proteins in lipid metabolism (Kostova et al., 2007), CFTRD508 conserved active site Cys of ubiquitin-conjugating enzymes (Morito et al., 2008), and the metastasis suppressor KAI1 (E2), of which there are more than 30 in mammals. E2s bind to (CD82) (Tsai et al., 2007). specific ubiquitin-protein ligases (E3s), which mediate the trans- gp78 has a complex domain structure for a single subunit E3. fer of ubiquitin to primary amines on substrates or to growing In addition to its RING finger, it has at least three more C-terminal chains of ubiquitin (polyubiquitin or multiubiquitin). In many domains in its extended cytoplasmic tail (Figure 1A). Each of cases, the E3s also undergo auto- or self-ubiquitylation. these is implicated in ubiquitylation and degradation of ERAD There are more than 500 E3s in mammals that can be divided substrates. They include a ubiquitin-binding CUE (coupling of into two major classes. The HECT E3s include 30 E3s and are ubiquitin conjugation to ERAD) domain and a C-terminal binding

674 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. Molecular Cell gp78 Ube2g2-Binding Region: Structure and Function

Figure 1. The gp78 G2BR and Its Interactions with Ube2g2 by NMR (A) Schematic representation of gp78 in the ER membrane (left). To the right is a linear representation of gp78 cytoplasmic tail with amino acids (corresponding to the entire human gp78) indicated. Peptides used in this study corresponding to the G2BR, specific mutations/truncations, and a scrambled (Scr) peptide control are shown below. Mutations are indicated in lowercase. (B) Overlay of 15N-HSQC NMR spectra of Ube2g2 in free (red) and G2BR-bound (blue) form. (C) The contact residues observed in an intermolecular NOESY spectrum of Ube2g2:G2BR are painted blue on the free Ube2g2 structure (PDB entry 2CYX). Region of E1 and E3 binding based on other E2-E3 pairs is indicated by the bracket. The active site Cys (C89) is in red. (D) 15N-HSQC of isotopically labeled G2BR in free form (red) and bound to Ube2g2 (blue). site for p97. Unique to gp78 is a high-affinity binding site for its that part of the complex is losing some degree of freedom and cognate E2, Ube2g2. This Ube2g2-binding region (G2BR) is is likely folding into a regular structure. required for the function of gp78 in cells (Chen et al., 2006). We now report that the G2BR binds Ube2g2 through an ‘‘Backside Binding’’ to Ube2g2 Induces G2BR Folding extended interface distinct from sites of RING finger and E1 To identify the binding surface between Ube2g2 and G2BR, we binding. This results in subtle changes in the Ube2g2 core that examined the interaction by monitoring the NMR spectra of each are manifested in functional alterations in loading with ubiquitin molecule independently. First, the Ube2g2 preparation was vali- and a marked increase in affinity for the gp78 RING finger, which dated by confirming the resonance assignments for Ube2g2 is reflected in enhanced ubiquitylation. compared to a previous study (Briggman et al., 2005). A secondary structure analysis based on chemical shifts (Wishart RESULTS and Sykes, 1994) combined with distance information from a 15N-edited NOESY-HSQC spectrum confirmed that our The G2BR Is a High-Affinity Binding Site for Ube2g2 Ube2g2 preparation matched the reported crystal structure To evaluate the interaction between the gp78 G2BR (Figure 1A) (Arai et al., 2006). Titration of G2BR into isotopically labeled and Ube2g2, we employed isothermal titration calorimetry (ITC) Ube2g2 (Figure 1B) yielded slow-exchange NMR spectra and

(Figure S1 available online). This confirmed the direct, high- indicated a Kd << 1 mM, consistent with the ITC data. An unex- affinity interaction of Ube2g2 with a 27 amino acid G2BR pectedly large number of the Ube2g2 HN-N peaks shifted in peptide. The 1:1 complex of E2 and G2BR has a dissociation the presence of G2BR, requiring reassignment. The 13C-edited constant (Kd)of21(±4)nM(Table 1). The reaction is exothermic, HSQC spectra of the methyl resonances of Ile, Leu, and Val resi- and the significant free energy change (DG=43.72 kJ mol1) dues (data not shown) indicated a localized binding site, which implies a very stable complex. The decrease in entropy reveals correlates with the largest shifts observed in Figure 1B. The

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Table 1. Dissociation Constants of E2-E3 Interactions 1 1 1 1 Complex Kd DH (kJ mol ) DS(JK mol T ) Method NMR Exchange Timescale Ube2g2:G2BR 21 (± 4) nM 56.3 (± 0.01) 41.8 (± 0.04) ITC slow

Ube2g2:G2BRDN 740 (± 110) nM 9.80 (± 0.40) 83.6 (± 1.43) ITC slow a Ube2g2:G2BRDC 192 (± 22) mM n.d. n.d. NMR fast

Ube2g2:G2BRM4-1 55 (± 18) mM n.d. n.d. NMR fast

Ube2g2:G2BRM4-2 9.5 (± 4) mM n.d. n.d. NMR fast Ube2g2:gp78-RING 144 (± 10) mM n.d. n.d. NMR fast

(Ube2g2:G2BRDN):gp78-RING 29 (± 5) mM n.d. n.d. NMR fast (Ube2g2:G2BR):gp78-RING 3 (± 1) mM n.d. n.d. NMR fast a n.d. = not determined.

secondary structure based on chemical shifts of Ube2g2:G2BR conserved secondary structural elements include a1 = T4–L18, was identical to free Ube2g2, and NOESY spectra of b1 = G23–P28, b2 = E36–M42, b3 = V53–S59, b4 = K70–F73, Ube2g2:G2BR exhibited equivalent patterns of connectivities 310 helix = S91–L93, a2 = V116–A128, a3 = V138–D146, and between HN-HN,HN-methyl, and methyl-methyl protons a4 = R148–L163. Strands b1–b4 form an antiparallel b sheet compared to free Ube2g2, implying that the overall structure of (Figures 2A and 2B). A dynamic region in the b4a2 loop is present Ube2g2 did not change significantly. between residues H94 and W110. This corresponds to an acidic The binding surface was determined by chemical shift extension (aa 96–108) that, in mammals, is limited to Ube2g2 and mapping on Ube2g2 and by intermolecular NOESY experiments. two other E2s (Ube2g1 and Ube2r1 [Cdc34 in yeast]). The back- Chemical shift mapping for Ube2g2 indicates the primary binding bone root-mean-square deviation (rmsd) between the free and site is on b strands b1–b3(Figure S2). In addition, the C-terminal bound Ube2g2 is 1.8 A˚ over all residues and decreases to 0.9 A˚ regions of helices a1 and a4 are perturbed by G2BR binding. if residues 96–108 in the b4a2 loop are excluded. Intermolecular NOESY cross-peaks were observed between Key structural changes were observed in Ube2g2 near the active G2BR and the methyl and amide protons of Ube2g2 (Figure S3) site upon binding the G2BR. Access to the active site Cys (C89) is that correspond to the surface indicated in blue on unbound via a channel flanked on either side by b4a2 loop and the a2a3loop Ube2g2 in Figure 1C. These NOEs refined the contact surface (Figure 2A). The b4a2 loop was observed to exhibit dynamic to residues V25, A26, E31, E38, L40–M42, E45, E50, F51–V53, behavior for aa 96–108 in the crystal structure of free Ube2g2 V159, L163, and L165. This surface includes the hydrophobic (Arai et al., 2006). In fact, there were three molecules in the asym- patch formed by V25, A26, L40–M42, and F51–V53 that is mostly metric unit, and the major difference between the molecules was conserved in other E2s (Hamilton et al., 2001; Moraes et al., 2001; the conformation of the b4a2anda2a3loops(Figure 3A). These Brzovic et al., 2006). The G2BR-binding site also includes nega- conformations are ordered; however, they exhibit higher B factors tively charged residues flanking the hydrophobic core. From the than the rest of the structure (Figure S2). The dynamic region of the 15 perspective of the G2BR, N-HSQC spectra (Figure 1D) of b4a2 loop contains a short 310 helix and generally extends away a biosynthetically prepared G2BR exhibited poor chemical shift from the protein in the three conformations in the free form; dispersion, consistent with a random coil conformation. The however, in the Ube2g2:G2BR complex presented here, the 310 backbone chemical shifts were assigned in the free G2BR helix is gone, and a region of the loop (P100–Y103) orients back peptide, and, except for indications of a nascent 4 residue helical toward C89. The multiple conformations of these loops, which turn at V583–K586, the peptide lacks any regular secondary appear to shift to a new average state in the Ube2g2:G2BR structure. The spectrum of G2BR in the presence of Ube2g2 complex, and the high B factors inthe crystal structures are consis- exhibits a dramatic increase in spectral dispersion and reso- tent withdynamic averaging as suggested by chemical shift effects nance linewidths consistent with a 1:1 complex of Ube2g2:G2BR observed in solution (Figure S2). The present structure may repre- (Figure 1D). This suggests that the G2BR must fold into a compact sent a low-energy population of the average, in which the proximity regular secondary structure utilizing all 27 residues to fit on the of Y103 and C89 changes from 20.8 A˚ in unbound Ube2g2 to 3.8 A˚ defined E2-binding site. Based on this, we redefine the G2BR in the complex. In addition, the a2a3 loop (comprising N131–G135) as amino acids 574–600 of gp78 instead of 579–600 as originally approaches C89 inthe complex. Thisinwardconformation isstabi- defined (Chen et al., 2006). lized in the 1.8 A˚ crystal structure by a new network of water-mediated hydrogen bonding, including Y103, C89, and Y83, as well as The Ube2g2:G2BR Crystal Structure Reveals Distant a network including E133 and S91. The structural modifications of Changes that Impact E2 Loading of Ubiquitin these two loops decrease accessibility around the active site The Ube2g2:G2BR complex was crystallized, forming rod- (compare Figure 1Cto4BandFigure 3B to 3C). These changes shaped crystals (0.1 mm 3 0.3 mm 3 0.1 mm). These diffracted are accompanied by an altered orientation of the C89 side chain to 1.8 A˚ resolution (Table 2). The structure shows that the from pointing toward the b4a2 loop in the unbound form to pointing Ube2g2 backbone is largely unchanged in the presence of away from the loop (80% probability) with bound G2BR G2BR (Figure 2A), as predicted by NMR measurements. The (Figure 2A and compare Figure 3Bto3C).

676 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. Molecular Cell gp78 Ube2g2-Binding Region: Structure and Function

Table 2. Data Collection and Refinement Statistics Y103 is buried and is not visible. These structure-based models predict direct consequences for loading of G2BR-bound Data Collection Ube2g2 with ubiquitin from E1. This was tested by comparing Space group P2 2 2 1 1 1 ubiquitin loading of Ube2g2 in the presence of either a control Cell dimensions scrambled peptide (Scr) (Figure 1A) or the G2BR. The G2BR a, b, c (A˚ ) 48.92, 60.15, 61.64 resulted in a marked decrease in the rate of E2 loading at a, b, g () 90, 90, 90 30C(Figure 3D) and almost no detectable loading at 12C Resolution (A˚ ) 50–1.8 (1.86–1.8)a (Figure S4). Deletion of the extended acidic portion of the Number of observations 105668 (5851) b4a2 loop (aa 96–108; Ube2g2D96–108), which is the major Number of unique reflections 16657 (1272) contributor to steric crowding around the active site, decreased the G2BR effect by almost 75%, and G2BR binding was retained bR 7.8 (43.7) merge (Figures 3D and S5). I/s 22.4 (2.7) Completeness (%) 95.4 (74.2) Ube2g2-Bound G2BR Forms an a Helix with Extensive Redundancy 6.4 (4.6) Contacts across the Backside of the E2 Refinement The crystal structure of Ube2g2:G2BR demonstrates that the Resolution (A˚ ) 43–1.8 (1.86–1.8) G2BR assumes a completely a-helical fold in the 1:1 complex c Rworking (%) 19.3 (23.1) and binds to a surface comprising the b sheet (b1–b3) and the d C termini of a1 and a4(Figure 2A), which is consistent with the Rfree (%) 24.1 (27.4) solution NMR data (Figure 1C). The buried surface at the inter- Number of atoms/B factors (A˚ 2) face of Ube2g2 and G2BR is 1950 A˚ 2. The entire peptide is Protein 1318/33.1 well ordered, and there is an extensive network of contacts Peptide 260/34.4 between the G2BR and Ube2g2, comprising both hydrophobic Water 164/40.7 and ionic or hydrogen-bonding contacts (Figures 2C, 2D, and Rmsd 2E and Table S1). The contact network is consistent with the Bond lengths (A˚ ) 0.005 thermodynamic data for formation of this complex and suggests Bond angles () 0.92 that the recognition is highly specific. In the G2BR a helix, there is Ramachandran plot evidence for 24 hydrogen bonds between all i and i+4 pairs, beginning with the Ser 574 carbonyl oxygen to Arg 578 NH and Most favored region (%) 87.7 running through Arg 596 carbonyl oxygen to Lys 600 NH. As Additional allowed region (%) 12.3 amino acids 574–600 extend completely across the backside Generously allowed region (%) 0 of Ube2g2, this suggests that extensions beyond this region Disallowed region (%) 0 do not interact with Ube2g2. This is in agreement with previous a Values in parentheses are for the highest resolution cell. data (Chen et al., 2006) and NMR studies of aa 574–643 of b Rmerge = Sj(I)j/s(I), wherein I is the observed intensity. gp78 (unpublished data). c R factor = ShkljjFojjFcjj / ShkljFoj, calculated from working data set. The presence of extensive contacts between Ube2g2 and d Rfree is calculated from 5% of data randomly chosen and not included in G2BR suggests that single-point mutations will not disrupt refinement. binding or activity. The initial characterization of the G2BR (Chen et al., 2006) indicated that the N- and C-terminal ends of the G2BR were each critical both for binding Ube2g2 and for The multiple states surrounding the active site seen in the the cellular function of gp78. To examine this observation, we as- crystal structures and the shift in conformational averaging in sessed binding of G2BRDN and G2BRDC (Figure 1A) to Ube2g2. the Ube2g2:G2BR complex in solution suggest that these G2BRDC binds Ube2g2 with relatively weak affinity (Kd = 192 changes may have an impact on interactions with E1 or ubiquitin [± 22] mM) (Table 1). The affinity between G2BRDN and Ube2g2 bound to C89. To further evaluate this, we compared the struc- is significantly stronger (Kd = 740 [± 110] nM) but nonetheless tures of Ube2g2 and Ube2g2:G2BR with two reported E2-Ub exhibits an 35-fold decrease in affinity compared to wild-type structures (Hamilton et al., 2001; Eddins et al., 2006). Superpo- G2BR. This confirms that the N- and C-terminal regions of sition of free Ube2g2 with the E2 component of Ubc1-Ub, which G2BR both contribute directly to the high-affinity binding forms K48 ubiquitin chains (Chen and Pickart, 1990), and observed with the intact domain. Binding of either G2BRDN or Ubc13-MMs2-Ub, which forms K63 chains (Deng et al., 2000) G2BRDC to Ube2g2 causes a subset of the observed chemical (Figure 3B), reveals that the C termini of either of the two bound shift changes seen with the G2BR, consistent with their pre- ubiquitins can readily be accommodated in the channel sur- dicted contacts (data not shown). rounding C89. However, superposition of Ube2g2:G2BR with On the basis of the Ube2g2:G2BR structure, we designed two the same two structures (Figure 3C) indicates that the area peptides with four mutations each to assess the most important around the active site becomes occluded due to rearrange- interactions, G2BRM4-1 and G2BRM4-2 (Figure 1A). These ments of residues M101–Y103 and E133–G135 (within the peptides were 15N labeled and examined by NMR (Figure S6). b4a2 loop and a2a3 loop, respectively), resulting in steric The 15N-HSQC spectra of both peptides indicated random coil clashes with the C termini of the two ubiquitins. In Figure 3C, conformations characteristic of wild-type G2BR. Titration of

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Figure 2. Crystal Structure of the Ube2g2:G2BR Complex (A) Ribbon representation of the superimposed Ube2g2:G2BR complex with free Ube2g2 (PDB entry 2CYX). G2BR is cyan, and Ube2g2 is light green in the complex. Free Ube2g2 is shown in orange. (B) Linear representation of the secondary structure and binding regions of Ube2g2. a helices are represented by open rectangles; b strands, by ﬁlled arrows; and the active site Cys, by a red dot. Binding regions are indicated below the sequence line as RING ﬁnger (magenta) and G2BR (green). The position of Ube2g2’s extended dynamic loop is indicated in orange. (C) Hydrophobic side chains of G2BR (light blue, residues in black) lock into the hydrophobic surface of Ube2g2 (dark green, residues in yellow). (D) Intermolecular hydrogen bonds and salt bridges between G2BR and Ube2g2 are shown. Side chains of G2BR are shown in blue; residues, in black; the Ube2g2 contact side chains and residues, in red. The P21 and I24 side chains of Ubeg2g2 were not displayed because the interaction is through backbone hydrogen bonds. (E) Contacts between Ube2g2 (green) and G2BR (blue) indicating residues involved in hydrogen bonds, salt bridges, and hydrophobic interactions. Black lines link each residue to its reciprocal contact, and X denotes G2BR residues that do not have direct contacts in the interface.

the gp78 RING finger-binding site (Zheng et al., 2000; Brzovic et al., 2003; Domi- nguez et al., 2004)(Figure 2A). To evaluate possible allosteric effects of G2BR on RING finger binding, the interaction of isotopically labeled Ube2g2 with gp78 RING finger was monitored by NMR (Figure 4). The gp78 RING finger- binding interface on Ube2g2 consists of the N-terminal end of a1, the b3b4 loop (F62–P69), and part of the b4a2 loop (W110–S115) (see Figures 2A and 2B for secondary structure elements). The solution structure of the gp78 RING finger is similar to other RING finger structures (R.D. and R.A.B., unpublished

G2BRM4-1 and G2BRM4-2 with unlabeled Ube2g2 yielded Kdsof data), and a reverse labeling experiment using isotopically 55 (± 18) mM and 9.5 (± 4) mM, respectively (Table 1). These labeled gp78 RING finger confirmed that the gp78 RING finger findings, combined with the data for G2BRDN and G2BRDC, side of the binding surface is similar to other RING finger:E2 underscore the highly distributed nature of the contacts in interactions. The interaction exhibited fast exchange

Ube2g2:G2BR binding. (Figure 4A) and a Kd of 144 (± 10) mM(Table 1 and Figure S7). This is consistent with the low affinity of many RING finger:E2 The G2BR Enhances the Affinity of Ube2g2 interactions (Lorick et al., 2005; Christensen et al., 2007). The for the gp78 RING Finger affinity of the gp78 RING finger for the Ube2g2:G2BR complex The positioning of the Ube2g2:G2BR interface on the backside was also determined. While the binding interface was of Ube2g2 suggests that this interaction should not present unchanged and remained in fast exchange, strikingly, the Kd a direct steric impediment to the interaction of Ube2g2 with decreased by 48-fold to 3 (± 1) mM(Table 1 and Figure S7).

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Figure 3. G2BR-Induced Structural Changes in Ube2g2 Occlude the Area around the Active Site and Correlate with Ubiquitin Loading (A) Superposition of active site region (full structures shown in small inset) for four Ube2g2 molecules (yellow, cyan, and magenta from the ligand- free Ube2g2 in PDB entry 2CYX and green in Ube2g2:G2BR) showing the conformational ﬂexi- bility of the b4a2 loop. (B and C) Images showing the surface rendering of Ube2g2 and Ube2g2:G2BR combined with the potential orientation of ubiquitin chains, based on known E2-Ub structures. Ube2g2 was superimposed onto the E2 coordinates of two published E2-Ub structures: Ubc1-Ub (PDB entry 1FTX, rmsd = 1.8A˚ ) and Ubc13-MMs2-Ub (PDB entry 2GMI, rmsd = 1.4A˚ ). We display only the surface of Ube2g2 and the C-terminal end of the ubiquitin molecule (in ball and stick form) for Ubc1-Ub (blue) and Ubc13-MMs2-Ub (magenta). In (B), the free Ube2g2 structure is rotated relative to the orientation of Figure 1C by 180 about the vertical axis and zoomed in on the active site. The active site Cys (C89) backbone is yellow, and its side chain is red. Some residues of Ube2g2 that play a role in the allosteric change are labeled in green. R74 of both ubiquitin tails are indicated; the C-terminal diglycine approaches C89. In (C), the G2BR-bound form of Ube2g2 is shown in an identical orientation as in (B). (D) 35S-labeled Ube2g2 or Ube2g2D96–108 generated by in vitro translation in E. coli lysate was incubated with 100 nM E1 and ubiquitin-lacking lysines (Ub K0), so as to avoid formation of polyubiquitin chains. The formation of thiolester-linked Ube2g2 (E2Ub) was assessed at 30C with saturating (4 mM) G2BR peptide or a control ‘‘scrambled’’ peptide (Scr). Shown is the average of two experiments for each condition. Rate constant, K, and 95% conﬁdence index are shown for each condition.

G2BR Enhances RING Finger-Dependent Ubiquitylation by Ube2g2 gp78 is a substrate for its own ubiquitylation in cells (Fang et al., 2001; Chen et al., 2006). To begin to assess the effect of the G2BR on ubiquitylation, we employed Separate binding experiments confirmed that the G2BR and a GST fusion of the cytoplasmic domain of gp78 (GST-gp78C) RING finger do not directly interact in the gp78 RING finger: in an autoubiquitylation assay (Lorick et al., 1999). Ube2g2 was Ube2g2:G2BR complex (data not shown). The allosteric rela- provided in 5-fold molar excess relative to glutathione Sephar- tionship of Ube2g2:gp78 RING finger affinity to Ube2g2:G2BR ose-immobilized GST-gp78C, and ubiquitylation of bead-bound association was tested by measuring the affinity of gp78 RING material was assessed. Ubiquitylation was totally dependent on finger for Ube2g2 in the presence of G2BRDN, which exhibits an intact gp78 RING finger (Figure 5A), and mutations of two an affinity for Ube2g2 intermediate between the full G2BR and key residues in the G2BR (L582S/L589S; gp78CL582,589S) signif-

G2BRDC. The measured Kd was 29 (± 5) mM(Table 1), which icantly decreased ubiquitylation. A truncated form of GST-gp78C indicates that even a smaller fragment of G2BR has effects lacking the G2BR (GST-gp78CD577–643) also demonstrated that translate through Ube2g2 and increase its affinity for the markedly diminished ubiquitylation (Figure 5B), consistent with gp78 RING finger. G2BR-dependent increased affinity of Ube2g2 for the gp78

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Figure 4. Interactions between gp78 RING Finger and Ube2g2:G2BR Complex (A) Ube2g2:G2BR complex was titrated with gp78 RING finger followed by acquisition of 15N-HSQC spectra of isotopically labeled Ubeg2g2 at each titration point. 15N chemical shifts in parts per million (ppm) are indicated on the y axis, and 1H chemical shifts in ppm are on the x axis. Three of the affected residues of Ube2g2 (N19, L66, and V113) are shown. The peaks shift from the free (blue) form toward the bound form (magenta) with addition of gp78 RING finger. The dissociation constant was determined by fitting the peak positions against ligand: protein concentrations. (B) The surface representation of Ube2g2:G2BR shows the G2BR interface (blue), RING interface (magenta), and active site cysteine (red).

RING finger. However, when G2BR peptide was provided in at least in vitro, with members of the UbcH5 (Ube2d1–3) E2 trans, there was a marked increase in ubiquitylation of truncated family (Lorick et al., 1999; Nadav et al., 2003; Kikkert et al., gp78 (Figure 5B). This rescue of activity was not observed with 2004). HsHRD1 and Trc8 lack identifiable regions that are anal- the G2BRDN, G2BRDC, G2BRM4-1, or G2BRM4-2 peptides (Figures ogous to the G2BR. Therefore, we asked whether the enhanced 5C and 5D). To determine whether the G2BR peptide also ubiquitylation observed with the G2BR was unique to the gp78 enhances ubiquitylation of heterologous proteins, we took RING finger. Addition of the G2BR to ubiquitylation reactions advantage of gp78’s function as an E4 in ubiquitylating proteins utilizing GST fusions of the C-terminal RING finger-containing already modified with a single ubiquitin (Morito et al., 2008). A regions of either HsHRD1 or Trc8 resulted in a marked increase fusion protein of ubiquitin and GFP with a C-terminal His6 tag in RING finger-dependent autoubiquitylation (Figure 5H, top, (Ub-GFP-His6) was ubiquitylated by GST-gp78CD577–643 lanes 5 and 8, and Figure S8). Ubiquitylation mediated by these as assessed using Flag-tagged ubiquitin (Figure 5E). As with E3s, as well as by GST-gp78CD577–643, was not increased by the gp78 autoubiquitylation, ubiquitylation of Ub-GFP-His6 by GST- G2BR when UbcH5B was used as the E2 (Figure 5H, bottom). gp78CD577–643 was substantially increased in the presence of Similarly, critical mutations in the G2BR do not decrease ubiqui- G2BR. Thus, the effect of the G2BR applies to ubiquitylation of tylation by UbcH5B (Figure S9). Thus, changes in the ERAD E2, a heterologous substrate, as well as to autoubiquitylation. Ube2g2, induced by binding of the G2BR at a site distant from To evaluate the effect of G2BR on substrate ubiquitylation where this E2 interacts with RING fingers, enhances ubiquityla- using the full-length gp78 cytoplasmic tail, gp78C and tion with at least three different mammalian E3s. This indicates gp78CL582,589S were compared. gp78C resulted in easily detect- both the specificity of the G2BR for Ube2g2 and its apparent able, high-molecular weight ubiquitylated Ub-GFP-His6, which effect on increasing productive interactions between Ube2g2 was not evident with gp78CL582,589S (Figure 5F). As and RING fingers. gp78CL582,589S showed some persistent autoubiquitylation in Figure 5A, these reactions were carried out by combining Increased Affinity of Ube2g2 for the gp78 RING Finger G2BR mutants with double mutations of Ube2g2 in key contact Accounts for the Enhanced Ubiquitylation points (Figure 5G; see Figure 2 and Table 1 for Ube2g2:G2BR To further evaluate the effect of the G2BR, we assessed the rate contacts). Though none of the double mutants of Ube2g2 of discharge of ubiquitin from Ube2g2, based on a previously substantially decreased ubiquitylation with either gp78C or used approach (Petroski and Deshaies, 2005), in the presence a single mutation (gp78CL582S), combining any of these E2 of saturating amounts of G2BR or an equal concentration of mutants with gp78CL582,589S resulted in a complete loss of ubiq- Scr. In these reactions, acceptors for ubiquitin are other proteins uitylation. Collectively, these findings demonstrate the functional in the reaction mix, including bacterial proteins and free ubiquitin importance of structurally defined key contact residues in the added in excess. In the absence of the RING finger, discharge of Ube2g2-G2BR interface. ubiquitin from Ube2g2Ub was slow and unaffected by G2BR peptide (Figure 5I). In the presence of 8 mM gp78 RING finger, G2BR Enhances Ubiquitylation by Ube2g2 loss of Ub-bound Ube2g2 was markedly accelerated. This was with Other RING Fingers significantly increased by saturating amounts of G2BR Ube2g2 also functions with other RING finger ERAD E3s (unpub- (Figure 5I). We wished to determine whether this apparent lished data). Two of these, HsHRD1 and Trc8, are implicated in increased rate of discharge can be accounted for by an human disease (Kostova et al., 2007). These E3s also function, increased population of Ube2g2:RING finger due to the

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presence of G2BR (<5% Ube2g2 bound with Scr; >50% bound even when the G2BR peptide is provided in trans to Ube2g2 with G2BR) or by as yet unknown allosteric effects on the together with the cytoplasmic domain of gp78 lacking the Ube2g2:gp78 RING finger complex mediated by the G2BR. G2BR sequence. Significantly, at least in vitro, the G2BR also Therefore, experiments were carried out with concentrations of increases ubiquitylation by heterologous ERAD RING finger gp78 RING finger at which approximately equal amounts of E3s. In the cell, this potential for cross-talk between different RING finger are bound to Ube2g2. Under these conditions, no ERAD ubiquitin ligases has the potential to contribute to associ- significant increase in discharge rate was observed in the pres- ations between ERAD RING finger E3s and, more importantly, ence of G2BR (Figure 5J). Thus, the G2BR-mediated increase to promote synergistic interactions among these proteins in affinity of Ube2g2 for the RING finger provides a molecular (Ye et al., 2005; Morito et al., 2008). Similarly, if the reported basis for the accelerated discharge of ubiquitin from gp78-oligomerization-dependent formation of K48 ubiquitin Ube2g2Ub and, by extension, for the observed enhancement chains on C89 of Ube2g2 occurs in cells (Li et al., 2009), this of ubiquitylation. will be facilitated by the enhanced affinity of Ube2g2 for the gp78 RING finger with G2BRs. This domain can serve both as DISCUSSION platforms for Ube2g2 bearing nascent ubiquitin chains and as a means to enhance transfer of ubiquitin from heterologous Structural Basis for the High-Affinity G2BR Binding Ube2g2s. to Ube2g2 In addition to the increase in RING finger binding, the The NMR and X-ray crystallographic determination of the G2BR- Ube2g2:G2BR structure, compared to apo-Ube2g2, reveals binding site on Ube2g2 provides an explanation for its high- a conformation in which there is narrowing of the channel projec- affinity interaction with gp78. The G2BR binds Ube2g2 through ting from the active site Cys89. This is largely due to reposition- an extended region of the core UBC domain of Ube2g2 that ing of the extended mobile acidic portion of the b4a2 loop, includes the C-terminal ends of both the a1 and a4 helices and including a dramatic alteration in orientation of side chains of extensive contacts with the intervening b sheets (b1–3). This M101 and Y103. All of the available structural data indicate backside binding substantially overlaps the noncovalent binding that this region is dynamically disordered (Arai et al., 2006; Li site for ubiquitin on UbcH5C (Brzovic et al., 2006). Whereas ubiq- et al., 2009; this study). One consequence of this repositioning uitin binds UbcH5C with a Kd of 300 mM, which is similar to the is a substantial delay in formation of Ube2g2Ub in the presence analogous binding of ubiquitin to Ube2g2 (R.D. and R.A.B., of G2BR. Depending on the rate limiting step(s) in vivo, slowing of unpublished data), the binding of the G2BR to Ube2g2 is of E2 loading with ubiquitin in the presence of the G2BR could allow higher affinity by > 10,000-fold. The basis for this difference is for ‘‘proofreading’’ by deubiquitylating enzymes in either sub- explained by the interacting interfaces. Ubiquitin, characterized strate selection or chain linkage. The region analogous to the by a stable, ordered structure, interacts with E2s through its b4a2 loop is important for interactions between other E2-E3 hydrophobic face centered on I44. The G2BR, on the other pairs (Lorick et al., 2005), as well as for Ube2g2:gp78 RING finger hand, folds from an unstructured state into a well-ordered a helix (this study). The acidic extension in this loop is also found in upon interacting with Ube2g2. At their interface, hydrophobic Cdc34. Mutations in acidic residues in this region in both side chains along the spine of the G2BR a helix interlock with Ube2g2 and Cdc34 translate into decreased RING finger-depen- Ube2g2 in a fashion similar to the UbcH5C:Ub complex. Addi- dent formation of K48-linked polyubiquitin chains (Li et al., 2007; tionally, charged and polar residues distributed along the Petroski and Deshaies, 2005). Moreover, Cdc34-Ub recently has

G2BR a helix on either side of the hydrophobic spine (Figures been shown to manifest 2-fold lower Kd for the core SCF E3 2C and 2D) form salt bridges and hydrogen bonds with than for Cdc34 (Saha and Deshaies, 2008). As the net effect of

Ube2g2, resulting in a high-affinity complex with a Kd of 21 nM. the G2BR, either in cells or in vitro, is to enhance ubiquitylation, potential effects of this dynamic loop on modulating transfer of Implications of Ube2g2:G2BR for Ubiquitylation ubiquitin from Ube2g2Ub, formation of K48 ubiquitin chains, and ERAD or in differential RING finger binding of Ube2g2Ub versus Until now, the existence of the G2BR as a high-affinity binding Ube2g2 may be of equal or greater significance than the site for Ube2g2 had been assumed to primarily provide a means pronounced inhibition of loading with ubiquitin that we observe. to increase the level of Ube2g2Ub in proximity to gp78 and to Addressing these possibilities and the unresolved question of enhance ubiquitylation (Chen et al., 2006). We have now uncov- whether ubiquitin can be transferred from E1 to Ube2g2 bound ered additional effects of the G2BR. This highly specific 27 aa either to the full cytoplasmic tail of gp78 or the isolated G2BR binding site for Ube2g2 has the striking effect of increasing the are important questions in ERAD and in E2 function that will affinity of Ube2g2 for the gp78 RING finger as a consequence require additional in vitro and cellular approaches. of conformational/allosteric changes in Ube2g2. The basis for the dramatic change in Kd, from 144 mMto3mM, is not yet fully Existence and Implications of E2-Binding Sites Distinct understood and awaits further structural studies. A striking from Ligase Domains consequence of this increased affinity is the increased associa- E2s interact with E1, as well as with HECT, RING, and RING tion of Ube2g2 with the gp78 RING finger, thereby facilitating the finger-like domains on E3s. The question now arises as to the discharge of ubiquitin from Ube2g2Ub through as yet unde- prevalence of other functionally significant E2 interactions. A fined RING finger-mediated effects (Ozkan et al., 2005; Petroski well-characterized example is the E2-like molecule Mms2 and Deshaies, 2005). This is reflected in enhanced ubiquitylation, (UEV1a), which binds Ubc13 through a region distinct from the

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682 Molecular Cell 34, 674–685, June 26, 2009 ª2009 Elsevier Inc. Molecular Cell gp78 Ube2g2-Binding Region: Structure and Function

G2BR-Ube2g2 interface and facilitates formation of K63-linked Ubc9 binding and Nup358/RanBP2 function (Pichler et al., ubiquitin chains (Eddins et al., 2006). Another example, 2004; Tatham et al., 2005). It will be of interest to know whether mentioned above, is ubiquitin, which plays a role in the proces- this second site of E2:E3 interaction has analogous effects to sivity of BRCA1-mediated ubiquitin chain formation, presumably those observed in this study. through alignment of multiple UbcH5CUb complexes (Brzovic It seems reasonable to postulate that E2-binding sites within et al., 2006; Christensen et al., 2007). The high-affinity interaction E3 complexes but distinct from canonical ligase domains may of Ube2g2:G2BR precludes backside ubiquitin binding in the be a property of a substantial fraction of E3s. To what extent context of Ube2g2 bound to gp78. However, it does not exclude these result in functionally significant allosteric effects on E2s, that alignment of multiple E2Ub complexes (not necessarily as demonstrated herein, now becomes an exciting area for Ube2g2) in vivo could occur in the context of a G2BR-bound future research. Ube2g2Ub and play a role in chain formation. There are several examples in which regions of E3s, which are EXPERIMENTAL PROCEDURES not included in their canonical ligase domains, bind E2s, e.g., Nedd4 and Ubr1p. For these, neither the sites of interaction on NMR Spectroscopy NMR samples were prepared in 50 mM Tris, 2 mM TCEP (pH 7.5) buffer, and their respective E2s nor the functional effects of this binding experiments were performed at 25C. NMR spectra were acquired on 600 and are known (Madura et al., 1993; Hatakeyama et al., 1997). There 800 MHz Varian INOVA spectrometers equipped with triple-resonance is evidence that SCF E3s recruit Cdc34 through part of the gradient cryoprobes. Details of resonance assignments, binding titrations, C-terminal extension of this E2 (Wu et al., 2002). Yeast Ubc7p, and intermolecular NOESY experiments are provided in the Supplemental which is the ortholog of Ube2g2, is recruited by Cue1p to the Experimental Procedures available online. ER membrane to function with the ERAD E3s Hrd1p and X-Ray Diffraction Doa10p (Kostova et al., 2007). Recent findings suggest that Crystallization screens were carried out with a Phoenix robot (Art Robbins the 180 amino acid cytoplasmic domain of Cue1p activates Instruments). The crystals were flash-frozen in liquid N2 after a short soak in Ubc7p in vitro in a RING finger-independent manner through a cryoprotection solution. A native data set was collected at beamline 22-ID unknown mechanisms (Bazirgan and Hampton, 2008; Kostova of the Advanced Photon Source, Chicago. The structure was solved by molec- et al., 2009). Moreover, an 50 aa domain in Cue1p, with ular replacement. The initial Fo –Fc map revealed the electron density for the some sequence homology to G2BR, directly binds Ubc7p. This G2BR. Details of crystallization, data acquisition, structure solution, model domain is sufficient to activate ERAD by Hrd1p in vivo and stim- building, and structure refinement are provided in Supplemental Experimental Procedures. ulates ubiquitylation in vitro, analogous to the G2BR (Kostova et al., 2009), and thus may represent a yeast equivalent of the Isothermal Titration Calorimetry G2BR. ITC was carried out using a VP-ITC microcalorimeter (MicroCal LLC, North- A possible parallel to gp78 and Ube2g2 comes from the ampton, MA) at 25C. The typical experiment included injection of 25–27 SUMO E3 Nup358/RanBP2. This E3 binds the SUMO E2 aliquots (10 ml each) of 0.1–0.5 mM peptide solution into a 0.01–0.10 mM (Ubc9) through regions on Ubc9 analogous to interactions protein solution in the ITC cell (volume 1.4 ml), stirring at 300 rpm. Additional details are provided in Supplemental Experimental Procedures. between ubiquitin E3s and E2s. However, this E3 also contacts Ubc9 through a second region with similarity to the In Vitro Ubiquitylation, E2 Loading, and Discharge Ube2g2:G2BR interface (Reverter and Lima, 2005). As pointed Autoubiquitylation was carried out as described (Kostova et al., 2009; Lorick out by Reverter and Lima, mutations in this interface impact et al., 1999). Ubiquitylation of bacterially expressed purified Ub-GFP-His6

Figure 5. G2BR-Mediated Increase in Ube2g2:gp78 RING Finger Afﬁnity Results in Enhanced Ubiquitylation (A) Glutathione Sepharose-bound GST fusions of the entire gp78 cytoplasmic tail (aa 309–643; GST-gp78C), an inactivating mutation in the RING ﬁnger (GST-gp78CRM) or in the G2BR (GST-gp78CL582,589S), or GST alone were incubated for 90 min in the presence of Ube2g2 and E1. After washing, ubiquitylated bead-bound material was assessed by SDS-PAGE and immunoblotting with antiubiquitin. See Figure S10 for Coomassie blue stain of fusion proteins. (B) Glutathione Sepharose-bound GST-gp78C, a truncation at amino acid 577 at the beginning of the G2BR (GST-gp78CD577–643), or GST alone was incubated with or without G2BR peptide as indicated and assessed in (A). (C and D) (C) and (D) were carried out as in (A) using GST-gp78CD577–643 and the indicated synthetic or recombinant peptides. Recombinant wild-type G2BR peptide is indicated by an asterisk to distinguish from the synthetic wild-type peptide.

(E) Ubiquitylation of Ub-GFP-His6 was carried out under the indicated conditions. The asterisk denotes two control samples in which Ub-GFP-His6 was added + after the reaction was ﬁrst terminated by the addition of 10% SDS for 10 min followed by 8 M urea. Following addition of urea, Ub-GFP-His6 was puriﬁed on Ni Sepharose beads, and samples were resolved by SDS-PAGE.

(F) Ubiquitylation of Ub-GFP-His6 was carried out as in (E) for 60 min. (G) Ubiquitylation reactions were carried out with GST-gp78C or the indicated Leu to Ser mutations together with wild-type Ube2g2 or the indicated double mutations of Ube2g2. (H) GST fusions of the cytoplasmic tails of HsHRD1, Trc8, or truncated gp78 were incubated as in (A) with the indicated peptides and E2s. (I) 35S-labeled Ube2g2 generated as in Figure 3D was loaded with wild-type ubiquitin for 10 min followed by inactivation of E1 by NEM (5 mM). Discharge of ubiquitin from Ube2g2 (<100 nM), in the presence of either G2BR or Scr (4 mM) with or without gp78 RING finger (8 mM), was monitored by measuring the fraction of Ube2g2Ub remaining at each time point. Shown on the left is a representative experiment using gp78 RING finger. Plotted on the right is the average of three independent experiments; error bars represent SD. (J) Discharge experiments as in (I) were carried out for 2 or 5 min at the indicated concentrations of gp78 RING finger. Shown on the left are images demonstrating loss of ubiquitin from Ube2g2. The insert on the right summarizes data from discharge experiments from both (I) and (J). Discharge rate constants, K, are shown for each experiment.

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(500 nM) was carried out using glutathione Sepharose 4B bound GST- Chen, B., Mariano, J., Tsai, Y.C., Chan, A.H., Cohen, M., and Weissman, A.M. gp78CD577–643. Reactions were terminated by 2% SDS (final). After 10 min, (2006). The activity of a human endoplasmic reticulum-associated degradation samples were diluted with 8 M urea in 50 mM Tris (pH 7.4) to 0.1% SDS. Ub- E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc. 2+ GFP-His6 was isolated on Ni beads and eluted with SDS-PAGE sample buffer Natl. Acad. Sci. USA 103, 341–346. 35 after washing. S-labeled Ube2g2 was translated in the S30 T7 bacterial TnT Christensen, D.E., Brzovic, P.S., and Klevit, R.E. (2007). E2-BRCA1 RING system (Promega) and used at < 100 nM. In loading experiments, Ub K0 interactions dictate synthesis of mono- or specific polyubiquitin chain link- was used. For discharge of ubiquitin from E2, E2 was loaded with wild-type ages. Nat. Struct. Mol. Biol. 14, 941–948. ubiquitin for 10 min. Reactions were quenched with 5 mM NEM, and the buffer Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., was exchanged to 50 mM Tris (pH 7.4) with Ub (80 mM) and peptide (4 mM) with Pickart, C., and Chen, Z.J. (2000). Activation of the IkB kinase complex by or without purified gp78 RING finger (aa 313–393); discharge was monitored at TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique 25C. Additional details are provided in Supplemental Experimental Proce- polyubiquitin chain. Cell 103, 351–361. dures. Dominguez, C., Bonvin, A.M., Winkler, G.S., van Schaik, F.M., Timmers, H.T., ACCESSION NUMBERS and Boelens, R. (2004). Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis, and docking approaches. Structure Coordinates and structure factors of the Ube2g2:G2BR complex were depos- 12, 633–644. ited in the Protein Data Bank under accession code 3H8K. Eddins, M.J., Carlile, C.M., Gomez, K.M., Pickart, C.M., and Wolberger, C. (2006). Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis SUPPLEMENTAL DATA of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13, 915–920. Supplemental Data include Supplemental Experimental Procedures, 10 fig- Eletr, Z.M., Huang, D.T., Duda, D.M., Schulman, B.A., and Kuhlman, B. (2005). ures, and 1 table and can be found with this article online at http://www.cell. E2 conjugating enzymes must disengage from their E1 enzymes before E3- com/molecular-cell/supplemental/S1097-2765(09)00341-4. dependent ubiquitin and ubiquitin-like transfer. Nat. Struct. Mol. Biol. 12, 933–934. ACKNOWLEDGMENTS Fang, S., and Weissman, A.M. (2004). A field guide to ubiquitylation. Cell. Mol. Life Sci. 61, 1546–1561. 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