Mechanistic Insights Into Active Site-Associated Polyubiquitination by the Ubiquitin-Conjugating Enzyme Ube2g2

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Wei Lia,1, Daqi Tub,1,2, Lianyun Lia, Thomas Wollerta, Rodolfo Ghirlandoa, Axel T. Brungerb,3, and Yihong Yea,3

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and bDepartments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305

Edited by Aaron J. Ciechanover, Technion–Israel Institute of Technology, Haifa, Israel, and approved January 8, 2009 (received for review August 28, 2008) Lys-48-linked polyubiquitination regulates a variety of cellular pro- be subsequently transferred en bloc to a substrate (16), which offers cesses by targeting ubiquitinated proteins to the proteasome for a novel mechanism of polyubiquitination. degradation. Although polyubiquitination had been presumed to In this article, we study the mechanism of Ube2g2 active site- occur by transferring ubiquitin molecules, one at a time, from an E2 associated polyubiquitination mediated by the ER-associated active site to a substrate, we recently showed that the endoplasmic RING finger ligase gp78. Our biochemical and structural analyses reticulum-associated RING ﬁnger ubiquitin ligase gp78 can mediate reveal a mode of E3–E2 interaction that occurs within a large the preassembly of Lys-48-linked polyubiquitin chains on the catalytic gp78–Ube2g2 heterooligomeric complex. This interaction brings cysteine of its cognate E2 Ube2g2 and subsequent transfer to a multiple Ube2g2s into close proximity to form active site-linked substrate. Active site-linked polyubiquitin chains are detected in cells polyubiquitin chains. Given that Ube2g2 is a major E2 involved in on Ube2g2 and its yeast homolog Ubc7p, but how these chains are ERAD, our findings provide important insights into how misfolded assembled is unclear. Here, we show that gp78 forms an oligomer via ER proteins are polyubiquitinated for proteasomal degradation. 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a Results BIOCHEMISTRY gp78 oligomer can simultaneously associate with multiple Ube2g2 gp78 Contains 2 Oligomerization Sites, One of Which Is Located Within molecules. This interaction is mediated by a novel Ube2g2 surface the gp78 Cytosolic Domain. We demonstrated that gp78 can mediate distinct from the predicted RING binding site. Our data suggest that the assembly of polyubiquitin chains on the active site of Ube2g2 a large gp78–Ube2g2 heterooligomer brings multiple Ube2g2 mole- and subsequent transfer to a substrate. We also showed that gp78 cules into close proximity, allowing ubiquitin moieties to be trans- forms an oligomer that might be critical for E2 active site-associated ferred between neighboring Ube2g2s to form active site-linked polyubiquitination (16). gp78 is a multispanning membrane protein polyubiquitin chains. anchored to the ER membrane with its catalytic domain facing the cytosol (23, 24). To identify the domains responsible for gp78 crystallography ͉ endoplasmic reticulum-associated protein degradation ͉ oligomerization, we expressed Flag-tagged, full-length gp78 (Flag- gp78 ͉ polyubiquitin chain gp78) together with GFP-fusion proteins comprising either the N-terminal transmembrane segments (gp78N-GFP) or the C- ovalent attachment of the 76-residue ubiquitin to polypeptides terminal cytosolic domain (gp78C-GFP) of gp78 in cells. Immu- Cregulates the stability, localization, or activity of the modified noprecipitation experiments showed that both gp78N-GFP and proteins (1–3). This modification requires concerted actions of 3 gp78C-GFP coprecipitated with Flag-gp78, unlike the control GFP types of enzymes: an activating enzyme (E1) that forms a thioester protein (Fig. 1 A and B), suggesting that gp78 contains at least 2 linkage (designated herein as ‘‘ϳ’’) between its catalytic cysteine oligomerization sites, one in the transmembrane region and the and the carboxyl group of the Gly-76 in ubiquitin; a conjugating other in its cytosolic domain. enzyme (E2) that receives ubiquitin from an E1; and an ubiquitin We focused our study on gp78C because the transmembrane ligase (E3) that catalyzes the transfer of ubiquitin from the E2 segments of gp78 are dispensable for the ligase activity (16, 23). active-site cysteine to a substrate (4). To date, 2 E1s and dozens of Using a set of gp78C deletion mutants (Fig. 1C), we mapped the E2 enzymes have been identified in mammals, whereas the number region required for gp78C oligomerization to a segment of 77 amino of E3s is in the range of several hundreds (5–8). Many E3 enzymes acids downstream of the RING domain (Fig. 1D). Further deletion contain a RING finger domain that interacts transiently with a analyses identified the element responsible for gp78C oligomeriza- cognate E2 to mediate polyubiquitination reactions (4, 9–12). Ubiquitination often occurs in the form of a polymer whereby the C-terminal Gly-76 in an ubiquitin moiety is linked to a lysine residue Author contributions: W.L., D.T., T.W., R.G., A.T.B., and Y.Y. designed research; W.L., D.T., L.L., and R.G. performed research; T.W. contributed new reagents/analytic tools; W.L., D.T., in another ubiquitin molecule. It had been presumed that poly- T.W., R.G., A.T.B., and Y.Y. analyzed data; and D.T., A.T.B., and Y.Y. wrote the paper. ubiquitin chains are formed by conjugating ubiquitin moieties, one The authors declare no conﬂict of interest. at a time, first to a lysine residue in a substrate, and then to a lysine This article is a PNAS Direct Submission. in the previously-attached ubiquitin molecule. This appears to be the case for some E2s (13–15). However, we and several other Freely available online through the PNAS open access option. groups recently found that ubiquitin chains can also be preas- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3FSH). sembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its 1W.L. and D.T. contributed equally to this work. yeast homologue Ubc7p both in vitro and in vivo (16–19). These 2Present address: Department of Cancer Biology, Dana–Farber Cancer Institute, Harvard Med- ubiquitin-conjugating enzymes are associated with endoplasmic ical School, Boston, MA 02115. reticulum (ER) membranes to modify misfolded polypeptides that 3To whom correspondence may be addressed. E-mail: [email protected] or have been exported from the ER lumen in a process termed [email protected]. ER-associated protein degradation (ERAD) (20–22). Intriguingly, This article contains supporting information online at www.pnas.org/cgi/content/full/ polyubiquitin chains preassembled on the active site of Ube2g2 can 0808564106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808564106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 Fig. 2. Oligomerization of gp78C is required for Ube2g2 active site-linked polyubiquitination, but is dispensable for ubiquitin ligation activity. (A) Purified recombinant gp78C variants. (B) Polyubiquitinating activity of gp78C⌬L. Poly- ubiquitination reaction conducted with E1, Ube2g2, Flag-Ub, and the indicated Fig. 1. Oligomerization of gp78. (A) Schematic representation of the gp78 E3 ligases was analyzed under both nonreducing (ϪDTT) and reducing (ϩDTT) variants tested in B.(B) Both the cytosolic domain and the transmembrane conditions by immunoblotting. Arrows indicate the mobility shift for Ube2g2 segments of gp78 can interact with full-length gp78. Detergent extracts of 293T active site-linked ubiquitin chains upon treatment with DTT. * indicates cells transfected with the indicated plasmids were subjected to immunoprecipi- Ube2g2ϳUb thioester. Note that the ubiquitin chains synthesized by gp78C⌬L tation (IP) followed by immunoblotting (IB) with the indicated antibodies. Note (⌬L) exhibit a pattern different from that generated by GST-gp78C (GST-WT) on that the expressed gp78 proteins comigrate with IgG (*). WCE, whole cell extract. a nonreducing gel. (C) gp78C⌬L cannot synthesize ubiquitin chains on the (C–F) Mapping the region in the gp78 cytosolic domain that is necessary for its Ube2g2 active site. Ubiquitin chains synthesized in the absence (no E3) or pres- self-association. (C) Schematic representation of the gp78 variants tested in D.(D) ence of the indicated gp78C variants were analyzed by immunoblotting. As in B, except that plasmids expressing the indicated gp78 variants were ana- E2ϳUb(n), E2-linked ubiquitin chains. Note that the Flag antibody has a higher lyzed. (E) Schematic representation of the gp78 variants used in F.(F)AsinB, affinity for polyubiquitin chains than for monoubiquitin. (D) Purified recombi- except that plasmids expressing the indicated gp78 variants were analyzed. * nant MBP-gp78C variants. (E) Polyubiquitinating activity of MBP-gp78C⌬L. Poly- indicates a gp78 degradation product. ubiquitination reactions conducted with the indicated MBP-tagged gp78C variants were analyzed under both nonreducing (ϪDTT) and reducing (ϩDTT) conditions. Arrows indicate the mobility shift for Ube2g2 active site-linked ubiq- tion as a hydrophobic segment containing amino acids 419–448 of uitin chains upon treatment with DTT. (F) Ubiquitin chains synthesized by MBP- gp78, which is predicted to form a loop (Fig. 1 E and F). gp78C⌬L (MBP-⌬L) are not linked to Ube2g2.

A Monomeric gp78C Mutant Retains Ubiquitin Ligation Activity but Fails to Promote Ube2g2 Active Site-Associated Polyubiquitination. contain a dimerization site, we assumed that the oligomeric state of GST-gp78C might more closely resemble that of full-length gp78. To investigate the role of E3 oligomerization in Ube2g2 active Moreover, at least for gp78C⌬L, the GST tag had little effect on its site-associated polyubiquitination, we wanted to generate a mono- ligase activity. In contrast, the MBP tag significantly reduced its meric gp78 mutant. To this end, we made a loop-deleted gp78C activity, as judged by a single-round ubiquitin ligation assay (Fig. mutant lacking residues 411–448 (gp78C⌬L). Wild-type gp78C and ⌬ S2). We therefore chose GST-gp78C as a reference when studying gp78C L were purified as both GST- and maltose-binding protein the activity of the untagged monomeric gp78C⌬L mutant. As (MBP)-tagged proteins. Removal of the tag from gp78C caused the demonstrated, GST-gp78C stimulated the formation of Ube2g2 protein to become insoluble, whereas cleavage of the tag from active site-linked ubiquitin chains that could be converted to free ⌬ gp78C L yielded soluble material (Fig. 2A). Gel filtration and chains by the reducing agent DTT (Fig. 2B, lanes 13–16 versus lanes ⌬ analytical ultracentrifugation analyses showed that gp78C L was a 9–12) (16). Accordingly, these chains could be recognized by monomer regardless of whether or not it contained the MBP tag Ube2g2 antibody (Fig. 2C, lanes 2 and 5) or coprecipitated with (Fig. S1a). In contrast, MBP-gp78C formed large-size oligomers Ube2g2 under a nonreducing condition (Fig. S3). By contrast, (Ͼ670 kDa). As expected, GST-gp78C formed oligomers even gp78C⌬L was capable of promoting the synthesis of polyubiquitin larger than MBP-gp78C, presumably caused by the dimerization of chains, but the chains were not linked to the Ube2g2 active site as GST (Fig. S1b). their mobility on SDS/PAGE gel was not altered by DTT (Fig. 2B, Because the gp78 N-terminal transmembrane domains also lanes 1–8). Consistently, ubiquitin chains formed in the presence of

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808564106 Li et al. Downloaded by guest on September 26, 2021 Fig. 3. gp78C⌬L stimulates the formation of free diubiquitin but is defective in building Ube2g2-linked diubiquitin. (A) As indicated by the experimental scheme, the transfer of Flag-UbK48R from Ube2g2 to an Ube2g2ϳubiquitin thioester was monitored by immunoblotting with Ube2g2 (E2) and Flag antibod-

ies. * indicates a nonspecific band. (B) As indicated by the experimental scheme, BIOCHEMISTRY the transfer of Flag-UbK48R from Ube2g2 to free ubiquitin was monitored by immunoblotting with Ube2g2 and Flag antibodies. Fig. 4. The monomeric gp78C mutant cannot promote Ube2g2 self-assembly and is defective in polyubiquitination of Herpc. (A) gp78C promotes Ube2g2 self-assembly. Equal amount of Flag-tagged (F-Ube2g2) and untagged Ube2g2 gp78C⌬L could not be recognized by the Ube2g2 antibody (Fig. 2C, were mixed in the presence of either GST (control) or increasing amounts of lanes 3 and 6), nor did they coprecipitate with Ube2g2 (Fig. S3). The GST-gp78C (lanes 3–6). Flag-Ube2g2 and its associated proteins were immuno- polyubiquitin ladder generated by gp78C⌬L appeared to be unan- precipitated by anti-Flag agarose beads and analyzed by immunoblotting. Lane Ϸ 1 shows input (30% of Ube2g2 used in the binding experiment). (B) gp78C⌬L does chored chains, as judged by the size increment of 9 kDa, which not facilitate Ube2g2 self-assembly. As in A, except that both GST-gp78C (GST- corresponded to the size of Flag-ubiquitin. Consistent with this WT) and gp78C⌬L(⌬L) were tested at a single concentration. (C) gp78C⌬Lis interpretation, these chains could be rapidly disassembled by defective in Herpc polyubiquitination. Polyubiquitination reactions conducted isopeptidase T, a deubiquitinating enzyme that only disassembles with E1, Ube2g2, Ub, Herpc, and the indicated gp78 variants were analyzed under unanchored chains (25, 26). Together, these results indicate that the reducing condition by immunoblotting and Ponceau staining. * indicates a monomeric gp78C mutant cannot promote Ube2g2 active site- nonspecific band. (D) gp78C⌬L can interact with Herpc. The indicated GST-fusion associated polyubiquitination although it retains ligase activity. This proteins were immobilized on glutathione beads and incubated with purified conclusion was further validated by using MBP-tagged gp78C and Herpc (lanes 2–4). Bound proteins were eluted and analyzed by SDS/PAGE and Coomassie blue staining. Lane 1 shows Herpc input (30%). a corresponding loop deleted gp78C mutant (Fig. 2 D–F). We next used an established single-round ubiquitin turnover ⌬ assay to further examine the ligase activity of gp78C L. To this end, The Monomeric gp78C Mutant Does Not Promote Ube2g2 Self-Asso- Ube2g2 precharged with Flag-UbK48R (donor) was incubated with ciation and Is Defective in Substrate Polyubiquitination. Because the Ube2g2 precharged with untagged wild-type ubiquitin (acceptor). transfer of ubiquitin between Ube2g2ϳUb thioesters requires The UbK48R mutant lacks Lys-48 and therefore can only serve as multiple Ube2g2 molecules in close proximity, we tested whether a donor because ubiquitin chains synthesized by gp78C are linked gp78C oligomer could simultaneously bind multiple Ube2g2s. We exclusively via Lys-48 (16). As demonstrated (16), wild-type gp78C incubated Flag-Ube2g2 with untagged Ube2g2 in the presence or could catalyze the transfer of Flag-UbK48R from an E2 active site absence of gp78C, and then immunoprecipitated Flag–Ube2g2 by to a second Ube2g2ϳubiquitin thioester to form diubiquitin-linked Flag antibody. Immunoblotting showed that GST-gp78C promoted Ube2g2 (Fig. 3A, lanes 6–8 and 14–16). In contrast, the monomeric the coprecipitation of untagged Ube2g2 with Flag–Ube2g2 (Fig. gp78C⌬L mutant failed to form diubiquitin-linked Ube2g2 (Fig. 4A, lanes 3–6). In contrast, gp78C⌬L failed to do so, although it was 3A, lanes 9–12). Instead, it produced a small amount of unanchored capable of binding to Ube2g2 (Fig. 4B, lane 3). Thus, a gp78 diubiquitin. This was caused by the transfer of Flag-UbK48R to free oligomer can recruit multiple Ube2g2 molecules to form a large ubiquitin present in the reaction. The formation of such a diubiq- E3–E2 heterooligomer. uitin species has been used as an indicator of ligase activity for some To determine the role of gp78 oligomerization in substrate RING E3s, and the amount of diubiquitin generated depends on ubiquitination, we tested the ability of gp78C⌬L to polyubiquitinate the concentration of the ubiquitin acceptor (13, 27). Indeed, when Herpc, an in vitro model substrate of gp78C (16). We showed that the Ube2g2ϳFlag-UbK48R thioester was incubated with excess wild-type gp78C forms Lys-48-linked polyubiquitin chains on ubiquitin, gp78C⌬L could generate diubiquitin more efficiently Lys-61 in Herpc (16). Interestingly, under the same condition, than wild-type gp78C (Fig. 3B, lanes 9–12 versus 13–16). These data gp78C⌬L ubiquitinated Herpc at a significantly reduced rate, confirm that oligomerization of gp78C is required for charging the although it efficiently generated ubiquitin chains that appeared Ube2g2 active site with polyubiquitin. Our results also indicate that unanchored (Fig. 4C). The defect in substrate polyubiquitination having ubiquitin ligase activity is not sufficient to stimulate E2 was not caused by lack of substrate association because in a GST active site-associated polyubiquitination. pull-down experiment gp78C⌬L associated with Herpc similarly to

Li et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 wild-type gp78C (Fig. 4D). We therefore conclude that oligomerization of gp78 is also required for efficient polyubiquitination of some gp78 substrates.

Structure of Ube2g2 Bound with a gp78 G2BR Peptide. RING finger E3s usually interact with cognate E2s via the RING domain. However, gp78 was reported to contain a second E2 binding site named G2BR (24). To understand how gp78 interacts with Ube2g2 to assemble a functional E3–E2 heterooligomer, we determined the crystal structure of Ube2g2 bound to a 28-residue peptide corresponding to the G2BR domain of gp78. Crystals in space group P43212 were formed by using an Ube2g2–G2BR complex preassembled on a size exclusion column. Iterative model building and refinement produced a model comprising 334 residues of 2 Ube2g2 monomers and 21 residues of the G2BR peptide per asymmetric unit with Rfree ϭ 25.6% and R ϭ 21.6% to dmin ϭ 2.76 Å (Table S1). The quality of the electron density map was excellent except for Ube2g2 residues 96–107 and G2BR residues 580–581 and 600. The 2 Ube2g2 monomers in an asymmetric unit are essentially identical, with a rmsd of 0.82 Å over 165 C␣ atoms. However, only 1 Ube2g2 interacts with a G2BR peptide, which is likely a result of crystal packing as the binding site on the second Ube2g2 is involved in crystal contact formation. The structure of Ube2g2 in our complex superimposed well onto that of Ube2g2 solved in isolation with a rmsd of 0.97 Å out of 165 C␣ atoms (28). Ube2g2 forms a compact domain comprising an antiparallel ␤-sheet with 4 strands (␤1–␤4) and 4 ␣-helices (␣1–␣4; Fig. 5A). In the Ube2g2–G2BR structure, 21 consecutive residues of the G2BR peptide (from Arg-580 to Lys-600) form a helix, resting on an Ube2g2 surface formed by strands ␤1–␤3 and the terminal portion of the helix ␣4 (Fig. 5A). The interface between Ube2g2 and G2BR buries a total surface area of 1,474 Å2. The interaction is formed predominantly by side chains of hydrophobic residues. Specifically, 8 residues in G2BR (Leu-582, Arg-585, Leu-589, Leu-590, Ala-593, Arg-596, Phe-597, Leu-598) form a combined 21 contacts (defined by pairs of carbon atoms that are Ͻ3.9 Å apart) with Ube2g2 (Fig. 5B). Among them, residues Ala-593 and Phe-597 make particularly extensive interactions as they together create 9 specific contacts: Ala-593 inserts into a pocket formed by Met-42, Val-53, and Leu-163, whereas Phe-597 snuggly fills up a depression formed by Phe-51, Gln-160, Leu-163, and Leu-165 of Ube2g2 (Fig. 5C). On the Ube2g2 side, Leu-163 and Leu-165 together make 7 specific contacts with G2BR by using their ␦ carbons, and Met-42 and Val-25 each forms 3 contacts with G2BR. In addition, 2 intermolecular hydrogen bonds were ob- served: NE and NH2 of G2BR Arg-594 each form a H-bond with O of Ube2g2 Leu-163. Phe-51 of Ube2g2 also interacts with Arg-596 in G2BR through hydrophobic contacts to its CG and C atoms, but the charged head group of Arg-596 forms no salt bridges Fig. 5. The structure of Ube2g2 bound to the gp78 G2BR domain. (A) Overall and its CG atom also has relatively weak electron density, suggest- structure showing the Ube2g2–G2BR complex. Contoured at 3.5 ␴ around G2BR is a ing that this interaction does not contribute much to the affinity 2Fo Ϫ Fc ␴A-weighted annealed omit map omitting G2BR. (B) The contacts between between Ube2g2 and gp78 (see below). G2BR and Ube2g2. (C) A close-up view on the most critical contacts around A593 and To understand the relative geometry of G2BR and RING F597 of G2BR. (D) The geometry of G2BR binding compared with RING binding and binding to Ube2g2, we aligned the structure of Ube2g2–G2BR with the active site. RING domain (blue) of the c-Cbl–UbcH7 complex (Protein Data Bank that of UbcH7 in complex with a RING ligase by using the ID code 1FBV), and Ub (red) of the Mms2-Ubc13ϳUb covalent complex (Protein Data respective E2 as a template. The result reveals that the G2BR Bank ID code 2GMI) are docked onto the Ube2g2–G2BR complex based on E2 binding site is located opposite to the predicted RING binding structural alignments. Overlay matrices were determined by DALI. surface on Ube2g2 (Fig. 5D). Moreover, both the G2BR and RING binding sites are well removed from the Ube2g2 catalytic center. L165A, L163A double mutant was severely defective. Moreover, a G2BR–Ube2g2 Interaction Is Required for Ube2g2-Dependent Poly- quadruple mutant combining all 4 mutations was completely inac- ubiquitination. To understand the role of G2BR–Ube2g2 interac- tive and did not bind G2BR (Fig. 6A). As a result, the Ube2g2 tion in regulating Ube2g2 active site-associated polyubiquitination, quadruple mutant could not be recruited to a gp78C–Ube2g2 we generated Ube2g2 variants that contained either single or heterooligomer (Fig. 6B). These results indicate that Leu-163 and multiple amino acid substitutions on the newly-identified G2BR Leu-165 in the carboxyl terminus of Ube2g2 are more critical for binding surface. These mutants exhibited similar elution profiles to G2BR binding, whereas Met-42 and Phe-51 may play a minor role. wild-type Ube2g2 on a size exclusion column, suggesting that they The interaction between gp78C and Ube2g2 was further exam- were properly folded. Substituting Met-42 and Phe-51 with Ala had ined by surface plasmon resonance experiments. Injection of little effect on the Ube2g2–gp78 interaction (Fig. 6A), whereas the Ube2g2 onto a carboxymethyl dextran-coated gold surface immo-

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808564106 Li et al. Downloaded by guest on September 26, 2021 with an affinity similar to wild-type gp78C, as judged by pull-down experiments (Fig. S4), suggesting that the stable interaction of gp78 with Ube2g2 is largely mediated by the G2BR domain. As anticipated, in vitro ubiquitination experiments showed that the L165A L163A double and the quadruple mutants were defective in assembling Ube2g2 active site-linked polyubiquitin chains (Fig. 6E) and could not efficiently polyubiquitinate Herpc (Fig. 6F). By contrast, the M42A and F51A mutants were able to assemble Ube2g2 active site-linked polyubiquitin chains and they had normal polyubiquitinating activities toward Herpc. Thus, the high-affinity Ube2g2–G2BR interaction is required for both Ube2g2 active site-linked polyubiquitination and substrate polyubiquitination. We therefore conclude that the G2BR domain is crucial for assembling a stable E3–E2 heterooligomer to fulfill the polyubiquitination function of gp78. Discussion In this article, we show that gp78 contains at least 2 oligomerization sites, which lead to the formation of large-size gp78 oligomers. We further demonstrate that a gp78 oligomer can bring multiple Ube2g2ϳubiquitin thioesters into close proximity, which presumably makes Ube2g2-linked ubiquitin a pre- ferred acceptor for chain extension and thus promote E2 active site-linked polyubiquitination. It has been well established that RING finger E3s bind cognate E2s via the RING domain (4). This interaction, albeit weak, is essential for promoting the transfer of ubiquitin from E2s to

substrates. RING binding requires 3 residues located at the tips of BIOCHEMISTRY the L1 and L2 loops, which are generally conserved among E2s including Ube2g2 (29). Thus, we assume that gp78 may also have a weak interaction with Ube2g2 via its RING domain. In this context, it is intriguing that a recent study identified a second E2 binding domain named G2BR in gp78 (24). The G2BR domain dramatically enhances the affinity of gp78 to Ube2g2. By contrast, removal of the RING domain does not have any significant affect on the gp78C–Ube2g2 interaction. These features distinguish Ube2g2 from other E2s that only transiently interact with E3 ligases via the RING domain (29), and thus suggest that Ube2g2 may operate by a distinct mechanism. Indeed, the residues critical for G2BR binding (L163 and L165) are largely conserved among the Ube2g2 family members, but are absent from other E2s. Moreover, unlike some E3s, gp78 does not undergo ubiquitination by Ube2g2. The generally weak E2–E3 interactions via the RING binding site may be critical for efficient polyubiquitination of many substrates. E2s presumably need to dissociate from an E3-substrate complex before they can be recharged for the next round of the Fig. 6. Ube2g2–G2BR interaction is required for active site-linked polyubiquiti- transfer reaction because the RING binding site on E2s overlaps nation and substrate ubiquitination. (A) Interaction of Ube2g2 with gp78C is with the E1 interaction surface (30). A transient E2–E3 interaction mediated by the G2BR domain. GST-tagged gp78C immobilized on glutathione is therefore favored as it allows Ͼ1E2ϳubiquitin thioester to beads was incubated with the indicated Ube2g2 variants. The bound proteins efficiently cycle on and off from a relatively stable E3–substrate were analyzed by immunoblotting. (B) The Ube2g2–G2BR binding is required for complex and thus enhance processivity, which is commonly defined Ube2g2 self-interaction. As in Fig. 4A except that the indicated untagged Ube2g2 in the ubiquitin field as the number of ubiquitin molecules trans- variants were incubated with Flag–Ube2g2 in the absence (Ϫ) or presence (ϩ)of ferred in a single round of substrate–E3 interaction. However, GST-gp78C. (C and D) Surface plasmon resonance analyses of GST–gp78C– dynamic E2–E3 interactions may not be suitable for certain sub- Ube2g2 interaction. (C) Representative sensorgrams for the binding of Ube2g2 to strates, particularly for those with a high dissociation constant for GST-gp78C. (D) The relative binding of wild-type Ube2g2 (E2 WT) and the quadruple mutant (E2 quad.) to GST-gp78C. (E) Active site-linked polyubiquitination E3 enzymes, because it is conceivable that these substrates may be by Ube2g2 variants. The reactions were analyzed under a nonreducing condition released from a ligase before a polyubiquitin chain is fully built. The by immunoblotting. (F) Polyubiquitination of Herpc by Ube2g2 variants. As in E, fact that diverse ERAD substrates are modified by a relatively small except that the reaction also contains Herpc and that the reaction was analyzed number of ER-associated ubiquitin ligases suggests that substrate under reducing condition. recognition must be promiscuous and transient in nature. ERAD substrates may therefore require a special mechanism to promote polyubiquitination. We propose that its processivity may be achieved via oligomerization and enhanced E2–E3 interaction bilized with GST-gp78C resulted in a concentration-dependent (RING and G2BR binding regions). These features allow a gp78 response (Fig. 6C). Thermodynamic evaluation of the data showed oligomer to simultaneously bind multiple Ube2g2ϳubiquitin thio- that Ube2g2 bound GST-gp78C with a KD of Ϸ5 nM, whereas the esters and thus reduce the reliance on recycle of E2s for chain quadruple Ube2g2 mutant barely interacted with GST-gp78C (Fig. extension. Moreover, the formation of such large-size E3–E2 6D). Interestingly, a RING deleted gp78C mutant bound Ube2g2 heterooligomers favors assembly of ubiquitin chains on the Ube2g2

Li et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 active site. The transfer of preassembled ubiquitin chains from format Protein Complex Suite (Qiagen) on a Phoenix robot (Art Robbins Instru- Ube2g2 to a substrate may help to overcome the detrimental effect ments) identified condition G7 (0.1 M Tris, pH 7.5, 3 M Na Formate) as the only hit. of substrate dissociation on polyubiquitination. Crystals of Ube2g2/G2BR complex were grown by using hanging drop vapor Interestingly, it was recently suggested that UbcH5c may also diffusion at 20 °C. A total of 1.5 ␮L of the complex was mixed with equal volume need to be assembled into an ordered oligomeric complex to of mother liquor consisting of 2.8 M Na Formate, 100 mM Tris (pH 7.7), 5 mM DTT and equilibrated against 1 mL of mother liquor. Crystals were directly frozen out processively polyubiquitinate substrates. In this case, an ordered of mother liquor into liquid nitrogen. A native dataset was collected at SSRL 11-1 UbcH5c oligomer may be formed by using a so-called ‘‘back-side and recorded on a MarMosaic 325 CCD detector. Integration, scaling, and merg- ubiquitin binding surface,’’ which is also present in the SUMO E2 ing of the diffraction data were performed by HKL2000 (33). Ubc9 (31, 32). The noncovalent E2–ubiquitin interaction would allow 1 UbcH5c to interact with an ubiquitin molecule conjugated Structure Determination and Refinement. The structure was solved by molecular to the active site of a second UbcH5c (31). Structural comparison replacement using Phaser (34) with the human Ube2g2 structure (Protein Data suggests that the back-side ubiquitin binding surface, if existing in Bank ID code 2cyx) as a search model. Data in the resolution range 50 to 2.76 Å Ube2g2, overlaps with the G2BR binding site on Ube2g2 (Fig. S5). were used in both rotation and translation calculations, which gave an obvious Thus, the mechanism by which G2BR assembles Ube2g2 oligomers solution with significant contrast, resulting in 2 Ube2g2 molecules in the asym- 3⅐ Ϫ1 must be distinct from that used by UbcH5c. Nonetheless, an metric unit with a Matthews coefficient (VM) of 4.47 Å Da and a solvent ordered E2ϳubiquitin thioester assembly may provide a common content of 72.5%. The G2BR helix was built by using COOT (35) and its register was mechanism that may enhance the processivity of polyubiquitination guided by some of its well-resolved side-chain electron density. Because of by allowing multiple E2ϳubiquitin thioesters to act simultaneously additional electron density, 2 residues of the cloning artifact sequence were added at the N terminus of Ube2g2. The resulting model was refined by using CNS in a reaction. 1.2 (36). The refinement consisted of alternating rounds of torsion angle molec- Materials and Methods ular dynamics simulated annealing, individual restrained thermal factor refinement, and model building in COOT. Crystallization, Data Collection, and Processing. The G2BR peptide correspond- Additional methods are available in SI Text. ing to gp78 residues 574–601 (NH2-SADERQRMLVQRKDELLQQARKRFLNKS- COOH) was synthesized by Elim Biopharmaceuticals. Lyophilized peptide was ACKNOWLEDGMENTS. We thank N. Soetandyo (National Institute of Diabetes dissolved at 1 mg/mL in protein buffer (100 mM NaCl, 20 mM Hepes, pH 7.0) and Digestive and Kidney Diseases) for technical assistance and M. Krause and M. supplemented with 10% DMSO. G2BR (2.2 mg) was mixed with 8 mg of Ube2g2, Gellert (National Institute of Diabetes and Digestive and Kidney Diseases) for loaded onto a HiLoad 16/60 Superdex 75 gel filtration column. The eluted peak critical reading of the manuscript. This work was supported by the intramural containing both Ube2g2 and G2BR was concentrated to 8.4 mg/mL. research program of the National Institute of Diabetes and Digestive and Kidney Initial screens of crystallization condition were carried out by using the 96-well Diseases, National Institutes of Health.

1. Weissman AM (2001) Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 21. Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: The long road to destruction. Nat 2:169–178. Cell Biol 7:766–772. 2. Welchman RL, Gordon C, Mayer RJ (2005) Ubiquitin and ubiquitin-like proteins as multi- 22. Ye Y (2005) The role of the ubiquitin-proteasome system in ER quality control. Essays functional signals. Nat Rev Mol Cell Biol 6:599–609. Biochem 41:99–112. 3. Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in 23. Fang S, et al. (2001) The tumor autocrine motility factor receptor, gp78, is a ubiquitin endocytosis and signaling. Science 315:201–205. protein ligase implicated in degradation from the endoplasmic reticulum. Proc Natl Acad 4. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533. Sci USA 98:14422–14427. 5. Jin J, Li X, Gygi SP, Harper JW (2007) Dual E1 activation systems for ubiquitin differentially 24. Chen B, et al. (2006) The activity of a human endoplasmic reticulum-associated degrada- regulate E2 enzyme charging. Nature 447:1135–1138. tion E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad 6. Chiu YH, Sun Q, Chen ZJ (2007) E1–L2 activates both ubiquitin and FAT10. Mol Cell Sci USA 103:341–346. 27:1014–1023. 25. Amerik A, Swaminathan S, Krantz BA, Wilkinson KD, Hochstrasser M (1997) In vivo 7. Pelzer C, et al. (2007) UBE1L2, a novel E1 enzyme specific for ubiquitin. J Biol Chem 282:23010–23014. disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein 8. Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitin degradation by the proteasome. EMBO J 16:4826–4838. and ubiquitin-like proteins. Annu Rev Cell Dev Biol 22:159–180. 26. Reyes-Turcu FE, et al. (2006) The ubiquitin binding domain ZnF UBP recognizes the 9. Lorick KL, et al. (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)- C-terminal diglycine motif of unanchored ubiquitin. Cell 124:1197–1208. dependent ubiquitination. Proc Natl Acad Sci USA 96:11364–11369. 27. Ozkan E, Yu H, Deisenhofer J (2005) Mechanistic insight into the allosteric activation of a 10. Xie Y, Varshavsky A (1999) The E2–E3 interaction in the N-end rule pathway: The RING-H2 ubiquitin-conjugating enzyme by RING-type ubiquitin ligases. Proc Natl Acad Sci USA finger of E3 is required for the synthesis of multiubiquitin chain. EMBO J 18:6832–6844. 102:18890–18895. 11. Joazeiro CA, Weissman AM (2000) RING finger proteins: Mediators of ubiquitin ligase 28. Arai R, et al. (2006) Structure of human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/ activity. Cell 102:549–552. UBC7). Acta Crystallogr F 62:330–334. 12. Deffenbaugh AE, et al. (2003) Release of ubiquitin-charged Cdc34-S-Ub from the RING 29. Zheng N, Wang P, Jeffrey PD, Pavletich NP (2000) Structure of a c-Cbl–UbcH7 complex: domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Cell 114:611– RING domain function in ubiquitin-protein ligases. Cell 102:533–539. 622. 30. Eletr ZM, Huang DT, Duda DM, Schulman BA, Kuhlman B (2005) E2-conjugating enzymes 13. Petroski MD, Deshaies RJ (2005) Mechanism of lysine 48-linked ubiquitin-chain synthesis by must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like the cullin–RING ubiquitin–ligase complex SCF–Cdc34. Cell 123:1107–1120. transfer. Nat Struct Mol Biol 12:933–934. 14. Rodrigo-Brenni MC, Morgan DO (2007) Sequential E2s drive polyubiquitin chain assembly 31. Brzovic PS, Lissounov A, Christensen DE, Hoyt DW, Klevit RE (2006) A UbcH5/ubiquitin on APC targets. Cell 130:127–139. noncovalent complex is required for processive BRCA1-directed ubiquitination. Mol Cell 15. Jin L, Williamson A, Banerjee S, Philipp I, Rape M (2008) Mechanism of ubiquitin-chain 21:873–880. formation by the human anaphase-promoting complex. Cell 133:653–665. 32. Knipscheer P, van Dijk WJ, Olsen JV, Mann M, Sixma TK (2007) Noncovalent interaction 16. Li W, Tu D, Brunger AT, Ye Y (2007) A ubiquitin ligase transfers preformed polyubiquitin between Ubc9 and SUMO promotes SUMO chain formation. EMBO J 26:2797–2807. chains from a conjugating enzyme to a substrate. Nature 446:333–337. 17. Ravid T, Hochstrasser M (2007) Autoregulation of an E2 enzyme by ubiquitin-chain 33. Otwinowski Z, et al. (1988) Crystal structure of trp repressor/operator complex at atomic assembly on its catalytic residue. Nat Cell Biol 9:422–427. resolution. Nature 335:321–329. 18. Cao J, et al. (2007) Ufd1 is a cofactor of gp78 and plays a key role in cholesterol metabolism 34. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fast by regulating the stability of HMG-CoA reductase. Cell Metab 6:115–128. translation functions. Acta Crystallogr D 61:458–464. 19. Bazirgan OA, Hampton RY (2008) Cue1p is an activator of Ubc7p E2 activity in vitro and in 35. Emsley P, Cowtan K (2004) COOT: Model-building tools for molecular graphics. Acta vivo. J Biol Chem 283:12797–12810. Crystallogr D 60:2126–2132. 20. Hampton RY (2002) ER-associated degradation in protein quality control and cellular 36. Brunger AT (2007) Version 1.2 of the Crystallography and NMR system. Nat Protoc regulation. Curr Opin Cell Biol 14:476–482. 2:2728–2733.

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