Ubiquitin Intermediate: Insight Into the Formation of the Self-Assembled E2Ub Conjugates

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Crystal Structure of UbcH5bUbiquitin Intermediate: Insight into the Formation of the Self-Assembled E2Ub Conjugates

Eri Sakata,1,2,7 Tadashi Satoh,3,4,7 Shunsuke Yamamoto,1,7 Yoshiki Yamaguchi,1,4 Maho Yagi-Utsumi,1,5 Eiji Kurimoto,1,6 Keiji Tanaka,2 Soichi Wakatsuki,3 and Koichi Kato1,5,* 1Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan 2Laboratory of Frontier Science, Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan 3Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan 4Structural Glycobiology Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 5Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan 6Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan 7These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.str.2009.11.007

SUMMARY also reported to serve as a signal for protein degradation (Baboshina and Haas, 1996; Koegl et al., 1999). However, ubiq- E2 ubiquitin-conjugating enzymes catalyze the uitination was also shown to have nonproteolytic roles. Lys63- attachment of ubiquitin to lysine residues of target linked polyubiquitination functions in DNA repair and transcrip- proteins. The UbcH5b E2 enzyme has been shown tional regulation (Hershko and Ciechanover, 1998; Pickart and to play a key role in the initiation of the ubiquitination Eddins, 2004). Interestingly, it was recently shown that linear pol- of substrate proteins upon action of several E3 yubiquitin is also involved in transcriptional regulation (Rahighi ligases. Here we have determined the 2.2 A˚ crystal et al., 2009; Tokunaga et al., 2009). The formation and elongation of polyubiquitin chains are per- structure of an intermediate of UbcH5bubiquitin formed by the cooperation of E2 and E3 enzymes (Hershko and (Ub) conjugate, which is assembled into an infinite Ciechanover, 1998). More than 600 E3 enzymes with diverse spiral through the backside interaction. This active architectures have been indentified, whereas all E2 enzymes complex may provide multiple E2 active sites, contain a topologically conserved domain containing the cata- enabling efficient ubiquitination of substrates. lytic cysteine residue (Pickart, 2001). This catalytic residue of Indeed, biochemical assays support a model in which E2 enzymes forms a thioester bond with the C terminus of ubiq- the self-assembled UbcH5bUb can serve as a uitin and it is subjected to nucleophilic attack by the lysine bridge for the gap between the lysine residue of the residue of target proteins. The conserved HPN motif located in substrate and the catalytic cysteine of E2. the proximity of the cysteine is responsible for the stability of the oxyanion intermediate during ubiquitin transfer (Reverter and Lima, 2005; Wu et al., 2003; Yunus and Lima, 2006). INTRODUCTION A key question in ubiquitination is how E2-E3 complexes can deal with the various acceptor sites distributed on the substrate The covalent attachment of ubiquitin to target proteins regulates surface to perform multiple ubiquitination and/or polyubiquitin a variety of cellular events, including the progress of cell cycle, elongation. Previous studies revealed diverse ubiquitination DNA repair, transcriptional regulation, and apoptosis (Hershko schemes. In recent years, structural studies of E2 and E3 and Ciechanover, 1998; Pickart and Eddins, 2004). The conjuga- enzymes showed that dynamic conformational changes of E3 tion of ubiquitin is initiated by the activation of its C terminus by ligases help to bridge the gap between the substrate and the E1 in an ATP-dependent manner, followed by ubiquitin transfer catalytic residue (Capili and Lima, 2007; Duda et al., 2008; Dye to the catalytic cysteine of E2 enzymes. The energy stored in and Schulman, 2007; Huang et al., 1999; Mizushima et al., the E2Ub thioester is utilized to conjugate ubiquitin to target 2007; Verdecia et al., 2003). The structure of the Mms2- lysine 3-amino groups, in a process mediated by E3 ubiquitin Ubc13ubiquitin complex revealed the structural basis of ligases. The fate of ubiquitinated proteins depends on the type Lys63-linkage specific polyubiquitin chain formation (Eddins of modification that they have been subjected to. The best- et al., 2006). In this structure, the donor ubiquitin of the Mms2- understood case is the Lys48-linked polyubiquitin chain involved Ubc13ubiquitin complex contacted Mms2 in an adjacent in protein degradation by 26S proteasomes (Hershko and Cie- complex, presenting its Lys63 residue into the active site of the chanover, 1998). Polyubiquitin chains with other linkages were Ubc13. Furthermore, several E2 enzymes have been reported

Figure 1. Structural Comparison between the Activated and Apo E2 Enzymes (A) Ribbon representation of the UbcH5bUb conjugate structure with UbcH5b in cyan and ubiquitin yellow. The ester bond between UbcH5b S85 and ubiquitin G76 are shown with stick model. The electron density of the bond is shown at 1.2 s level by a mesh surface. (B) Superposition of UbcH5b (cyan) and apo UbcH5b (magenta, PDB code: 2ESK). (C) Molecular details of the covalent bond between UbcH5b and ubiquitin (Ub) with the HPN motif residues. Dashed lines indicate potential hydrogen bond interactions. (D) Superposition of UbcH5bUb (blue) and apo UbcH5b (PDB code: 2ESK) showed in pink. (E) Model for catalytic reaction of nucleophilic attack on UbcH5bUb. (F) Superposition of Ubc9 (green)-RanGAP1 (blue) SUMO1 (magenta) (PDB code: 1Z5S) and apo Ubc9 (pink) (PDB code: 2PE6). (G) Superposition of Ubc13 (green) Ub (yellow) (PDB code: 2GMI) and apo Ubc13 (pink) (PDB code: 1JAT). to interact with ubiquitin (Brzovic et al., 2006; Eddins et al., 2006; RESULTS Miura et al., 1999) and form the self-assembled E2Ub conjugates to enhance ubiquitination (Brzovic et al., 2006; Merkley Structural Determination and Overall Structure and Shaw, 2004; Varelas et al., 2003). of the UbcH5bUb Conjugate UbcH5b is one of the E2 enzymes that have been demon- The thioester-linked UbcH5bUb conjugate is a catalytically strated to form polyubiquitin chains in cooperation with several activated intermediate, and thus unstable and not suitable for E3 enzymes, including the Cullin-based E3 ligases, BRCA1, crystallization. In order to trap a less activated conjugate, the CHIP and E6AP (Brzovic et al., 2006; Sakata et al., 2007; Wind- catalytic residue Cys85 of UbcH5b was substituted with a serine heim et al., 2008; Wu et al., 2003). Recent studies showed that residue. Ubiquitin was enzymatically conjugated to UbcH5b the UbcH5 family catalyzes multiple ubiquitination of lysine resi- (C85S) by using E1, ATP and Mg2+. The resultant oxyester conju- dues of the substrate (Kirkpatrick et al., 2006), followed by gate was purified with cation exchange column chromatography Lys48-linkage specific elongation by the Ubc1 family (Rodrigo- and crystallized by the hanging drop method. The UbcH5bUb Brenni and Morgan, 2007; Windheim et al., 2008). UbcH5b has conjugate structure (PDB code: 3A33) was determined at 2.2 A˚ been postulated to gain access to various acceptor sites on the resolution by molecular replacement using the apo structures substrate in an efficient manner (Windheim et al., 2008), but the of UbcH5b (PDB code: 2ESK) and ubiquitin (PDB code: 1UBQ) structural basis of this mechanism remains largely unclear. as search models (Figure 1A and Table 1). The areas of the elec- Here we show the crystal structure of an UbcH5bUb conju- tron density map of the ester linkage between the introduced gate and present structural insights into the mechanism by which Ser85 (UbcH5b) and the C-terminal Gly76 (ubiquitin), as well as the self-assembled E2Ub conjugates enhances substrate the core UbcH5b and ubiquitin structures were very clear ubiquitination. (Figure 1A). The overall structure of UbcH5b and ubiquitin was

Table 1. Data Collection and Reﬁnement Statistics of UbcH5bUb Crystallographic Data

Space group P6122 Unit cell a / b / c (A˚ ) 72.5 / 72.5 / 176.6 a / b / g () 90.0 / 90.0 / 120.0 Data Processing Statistics Beamline PF BL5A Wavelength (A˚ ) 1.0000 Resolution (A˚ )a 50 - 2.20 (2.28-2.20) Total reflections 177 153 Unique reflections 14 779 Completeness (%)a 99.6 (97.2) a Rmerge (%) 7.0 (27.8) I / s (I)a 47.1 (4.6) Figure 2. Single Mutations in the HPN Motif Resulted in a Significant Refinement Statistics Defect in Ubiquitination Evaluation of E2 activities of the UbcH5b mutants in vitro autoubiquitination of ˚ Resolution (A) 20 – 2.20 MBP-Rma1. The UbH5b mutants with a single amino acid substitution in the

Rwork 23.1 HPN motif displayed E2 activities comparable to the wild-type.

Rfree 28.0 Root-mean-square deviation from ideal values approach of the 3-amino group lysine residue of substrates or ˚ Bond length (A) 0.013 the acceptor ubiquitin. The point mutation of these residues Angle distance (A˚ ) 1.46 impaired MBP-Rma1 autoubiquitination (Figure 2), and the Ramachandran plot (%) mutation of Asp117 resulted in a significant defect in ubiquitina- Most favored 90.6 tion (Figure 2). This residue corresponds to Asp127 in Ubc9 Additionally allowed 8.9 (Figure 1F), which was involved in the efficient SUMO-transfer Generously allowed 0.5 (Reverter and Lima, 2005; Yunus and Lima, 2006). The side chain of UbcH5b Arg90, which is involved in many Disallowed 0 interactions, including the contact through hydrogen bonds to Number of non-hydrogen atoms the main-chain oxygen of ubiquitin Gly35 and to the side-chain UbcH5b 1195 O31 of Gln40, is flipped out in the apo structure of UbcH5b in Ubiquitin 601 order to avoid the repulsion from ubiquitin Arg74 (Figures 1D Water molecules 62 and 3A). The mutation of Arg90 to glutamic acid but not to Average B-values (A˚ 2) alanine resulted in a ubiquitination defect (Figure 2), suggesting UbcH5b 36.5 that an unfavorable interaction between ubiquitin (Arg74) and the UbcH5b mutant (Glu90) may inhibit ubiquitin transfer to the Ubiquitin 33.9 substrate. Water molecules 37.6 a Values in parentheses are for the highest-resolution shell. The UbcH5bUb Conjugate Adopts a Transition State Structure In our structure, the conjugated ubiquitin was observed interact- similar to the apo structures in which both of molecules are not ing with the E2 active site. The UbcH5b Asn77 and Asp117 conjugated, and the Ca positions could be superimposed with interact with the GG motif at the C terminus of ubiquitin (Figures an rmsds of 0.60 A˚ and 0.36 A˚ , respectively (Figure 1B). This 1C and 3A). The Asn77 Nd, which has been considered to form shows that ubiquitin conjugation induces no drastic conforma- an oxyanion intermediate during the nucleophilic attack on the tional changes to either UbcH5b or ubiquitin, as seen in the thioester bond (Figure 1E) (Reverter and Lima, 2005; Wu et al., previous E2 structures of Ubc13Ub (Eddins et al., 2006). The 2003; Yunus and Lima, 2006), forms a hydrogen bond with the only conformational difference between the two UbcH5b struc- C-terminal Gly76 oxygen, while it turns away to form hydrogen tures was observed for the conserved HPN motif around the bonds with the main chains of Asn114 and Asp117 in the apo covalent bond with ubiquitin. Upon ubiquitin conjugation to structure (Figures 1C and 1D). Previous studies indicated that UbcH5b, a groove created by the loops containing Asn114 and E2Ub/ubiquitin-like protein (Ubl) conjugates might adopt Asp117 was exposed (Figure 1C and 1D). The accessible surface a similar configuration to arrange the target lysine residue toward area of Asn114 (69.7 A˚ 2) and Asp117 (89.6A˚ 2) of UbcH5bUb are the thioester bond (Reverter and Lima, 2005; Wu et al., 2003; considerably increased compared with those of apo UbcH5b Yunus and Lima, 2006). In the present structure, the relative (52.7 A˚ 2 and 29.2 A˚ 2, respectively) (see Table S1 available on- spatial arrangement of ubiquitin to E2 is different from those re- line). This exposed groove seems to be preferable for the ported previously, in which Ub or Ubl contacted not only the

Figure 3. Schematic Representation of the Binding Interface between UbcH5b and Ubiquitin Interaction diagram between UbcH5b and conjugated ubiquitin around the catalytic site (A) and symmetry- related ubiquitin in the backside surface against the catalytic site (B). Dashed lines indicate potential hydrogen bonds (black) and hydrophobic interactions (pink), respectively.

face of the b1-b3 backside surface against the catalytic cysteine residue covers a significant continuous surface, burying a total surface area of 802 A˚ 2 (Figure 5A). The UbcH5bUb conjugate is assembled into an infinite spiral in the crystal through this interaction (Figure 5B). On the other hand, the other two contact areas are relatively small (228 A˚ 2 [Ub1] and 517 A˚ 2 [Ub3]) (Figure 4A), suggesting that the crystallographic interaction is non-physiological. In fact, our nuclear magnetic resonance (NMR) analysis confirmed that only the interaction between the b1-b3 backside surface and ubiquitin is present in solution (Figures 4B–4D). The properties of the amino acid residues involved in this ‘‘backside interaction’’ include both extensive polar and nonpolar contacts through the canonical ‘‘Ile44 surface’’ of ubiquitin, which is a common binding site for various ubiquitin binding domains (Figure 3B and 5A). Our crystallographic data showed that Pro17, Ser22, Ala23 and Val49 form tight interactions with ubiquitin. NMR analysis of UbcH5b mutants revealed that substitutions of these residues compromised the backside interaction (data not shown). Especially, mutation of Ser22 to Arg impaired this interaction resulting in defects of autoubiquitination of MBP-Rma1 (Figure 6A). These data are consistent with previous studies of active site but also the a3 helix of E2 (Figure S1)(Hamilton et al., apo UbcH5c (Brzovic et al., 2006). The interaction mode of apo 2001; Reverter and Lima, 2005). However, the precise arrange- UbcH5c with ubiquitin, estimated from intermolecular nuclear ment at the catalytic site is consistent with the proposed model, Overhauser effect data, was appreciably overlapped on our in which the E2 channel coordinates the C terminus of ubiquitin crystal structure (Figure S2). These data imply that the conjuga- to promote nucleophilic attack (Figure 1C, 1D, and 1F) (Reverter tion of ubiquitin does not affect the backside interaction of and Lima, 2005; Wu et al., 2003; Yunus and Lima, 2006). On the UbcH5b with ubiquitin. In addition, sequence alignment of other hand, the relative orientation of ubiquitin to E2 is similar in UbcH5b revealed that these key residues such as Ser22 are the UbcH5bUb and Ubc13Ub conjugates (Eddins et al., conserved among the E2 enzymes Ubc2b and UbcH5c, which 2006). However, the catalytic site might rather adopt an inactive have been reported to interact with ubiquitin thorough the back- form, because the hydrogen bond between Asn79 Nd and the side surface (Figure S3)(Brzovic et al., 2006; Miura et al., 1999). C-terminal Gly76, necessary to stabilize the oxyanion interme- Although previous studies reported that the backside interac- diate, was not observed (Figure 1G). Taken together with the tion is necessary for the formation of the polyubiquitin chain results of our biochemical assay (Figure 2), we propose that our (Brzovic and Klevit, 2006), the underlying mechanisms that E2Ub structure shows anintermediate state for ubiquitintransfer. enhance ubiquitination remain to be fully understood. We found that the backside interaction of UbcH5bUb conjugate resulted The Self-Assembly of the UbcH5bUb Conjugate in the formation of a self-assembled complex in the crystal (Fig- through the Backside Interaction ure 5B). On the basis of this observation, we postulated that the In the crystal, UbcH5b was found to contact three ubiquitin self-assembled conjugates may bridge the space between the molecules of the neighboring UbcH5bUb (Figure 4A). The inter- catalytic cysteine of E2 and E3. To examine this possibility, we

Figure 4. NMR Analysis of the Interaction between UbcH5b and Ubiquitin (A) Packing interaction between UbcH5b and three ubiquitin molecules in the crystal lattice. The UbcH5bUb conjugate and the symmetry-related three conjugates are shown as surface and cartoon representations, respectively. The Ub2 involved in signiﬁcant interaction through the canonical ‘‘Ile44 surface’’ is high- lighted as magenta. (B) Chemical shift perturbation data for the UbcH5b and monoubiquitin interaction. 1H-15N HSQC spectrum of [15N] UbcH5b in presence (red) and absence (black) of ubiquitin. (C) NMR chemical shift perturbation data for UbcH5b upon binding to ubiquitin. The data are displayed for each residue of UbcH5b according to the equation 2 2 1/2 [(0.2dN) + dH ] , where dN and dH represent the change in nitrogen and proton chemical shifts upon mixing with ubiquitin. Secondary structures are shown beneath the residue number of UbcH5b. (D) Mapping of the perturbed residues of UbcH5b upon binding to ubiquitin. The perturbed residues are colored in red and the color gradient indicates the strength of the perturbation. Proline residues are shown in gray. Ribbon diagrams are provided in the same orientation with surface diagrams of Figure4A and 4D (left).

Figure 5. Formation of the Self-Assembly of the UbcH5bUb Conjugate through the Backside Interaction (A) Side-chain interaction at the binding interface. The structure of UbcH5b and ubiquitin are colored in cyan and yellow, respectively. Side chains involved in the backside interaction are shown in stick representation. (B) UbcH5b in the crystal depicting a backside interaction with ubiquitin. UbcH5bUb forms an inﬁnite spiral in the crystal. A unit interface of the backside interaction was surrounded by dashed-line box.

designed an autoubiquitination experiment of MBP-Rma1 as side interaction, ubiquitination by UbcH5b(F62A) would be a model E3 (Matsuda et al., 2001) by combined use of enhanced in the presence of this conjugate. The biochemical UbcH5bUb and a UbcH5b mutant with impaired E3-binding. analysis of MBP-Rma1 suggested that UbcH5b(F62A) The phenylalanine at the b3-b4 loop of UbcH5b was shown to possesses very weak ubiquitination activity in comparison with be necessary for binding to E3 ligases (Huang et al., 1999; Zheng wild-type UbcH5b (Figure 6A). Increasing amounts of the et al., 2000). In fact, the mutation of Phe62 to Ala in UbcH5b UbcH5bUb conjugates with the ester linkage were mixed impaired the interaction with MBP-Rma1 (Figure S4) and con- together with wild-type UbcH5b or UbcH5b(F62A) in the pres- sequent autoubiquitination (Figure 6A). In contrast, the ence of E1, ubiquitin and MBP-Rma1. Interestingly, ubiquitina- UbcH5bUb conjugate with the ester linkage retained the ability tion by UbcH5b(F62A) was facilitated by the addition of the cata- to bind E3 but lacked the activity to transfer ubiquitin to lytically inactive UbcH5bUb conjugate, whereas such substrates (Figure 6A). Therefore, if the UbcH5bUb conjugate enhancement was not observed for wild-type UbcH5b (Fig- has a UbcH5b-recruitment ability onto the E3 through the back- ure 6B). In either case, excess of the conjugate inhibited

ubiquitination, probably because a highly assembled complex of of ubiquitin to lysine residues exposed on its surface. Recently, it the inactive E2Ub conjugates precluded the access of the was reported that in human cells UbcH10 preferentially functions active E2 complex to lysine residues of the substrate. Consis- as a partner of APC E3 ligase instead of the Ubc4/5 family tently, the introduction of the S22R and F62A mutations, impair- members (Summers et al., 2008). UbcH10, which possesses ing both E3 binding and the backside interaction, resulted in the several conserved residues of the backside interaction, may complete abrogation of the E2 activity (Figure 6A). These data form the self-assembled E2Ub conjugates to facilitate multiple indicate that the catalytically inactive UbcH5bUb conjugates ubiquitination of substrates. facilitate the recruitment of the active E2 complex and thereby In contrast, there are several E2 enzymes that form polyubiqui- enhance substrate ubiquitination. tin chains with a specific linkage. These enzymes possess a ubiquitin-interacting site distinct from the backside surface, or DISCUSSION couple with another ubiquitin-binding protein for guidance of the acceptor ubiquitin to present a specific lysine residue to The Key Residues for the Backside Interaction the thioester bond (Eddins et al., 2006; Kim et al., 2009; Petroski Are Conserved among Several E2 Enzymes and Deshaies, 2005). The crystal structure of the UbcH5bUb conjugate provides structural insights into the self-assembly of an E2Ub conjugate Bridging the Gap by the Self-Assembled E2Ub with backside interaction. We revealed that the key residues Conjugates involved in the backside interaction, i.e., Pro17, Ser22, Ala23, Structural studies of E3 have revealed a gap between the and Val49, are conserved among ubiquitin-interacting E2 substrate and the catalytic cysteine of E2 docked on E3 (Huang enzymes (Figure S3). It was found that Ube2e2, Ube2e3, et al., 1999; Zheng et al., 2002; Zheng et al., 2000). For example, Ube2g1, Ube2g1, and UbcH10 possess these conserved resi- there is a 50 A˚ gap between the substrate and the catalytic dues in the backside surface. These E2 enzymes may potentially cysteine of E2 docked on the SCF complex (Schulman et al., interact with ubiquitin through the backside surface and form the 2000; Zheng et al., 2002). Recent studies have revealed several self-assembled E2Ub conjugates. mechanisms underlying polyubiquitin chain formation by E2 and These corresponding residues for the backside interaction E3 enzymes. A significant conformational change of Rbx1, may also act as structural determinants governing E2 specific- induced by the conjugation of Nedd8, allows E2 to locate in ities of Ubls. We previously reported that Nedd8 interacts with the proximity of the target residue (Duda et al., 2008). The struc- UbcH5b, but not with the Nedd8 E2, Ubc12 (Sakata et al., tural flexibility of the polyubiquitin chain may accommodate the 2007). Intriguingly, it was recently suggested that the backside spatial constrains (Ryabov and Fushman, 2006). Recently, it surface of Ube2g2 was recognized by the G2BR domain of was reported that the allosteric conformational change near gp78, an E3 ligase (Das et al., 2009; Li et al., 2009). These notions the catalytic site of Ube2g2 was induced upon binding with the imply that the backside surfaces of the E2 enzymes display crit- E3 ubiquitin ligase gp78, resulting in the enhancement of ubiqui- ical determinants of selective recruitment of E2 enzymes to the tination (Das et al., 2009; Li et al., 2009). E3 ubiquitin ligase. In addition to these conformational flexibilities, we propose that the E2Ub conjugate forms a spiral complex containing Structural Basis for the Multiple Ubiquitination multiple active sites that can bridge the gap between the lysine Interestingly, the assembled UbcH5bUb conjugates adopts an residues of target protein and E3 ubiquitin ligase. Our biochem- intermediate configuration in which Asn77 Nd forms a hydrogen ical data underline the significance of a ‘‘riding-on’’ mechanism, bond with the C-terminal Gly76 oxygen to stabilize the transition in which the self-assembled E2Ub conjugates act as a bridge state. This implicates that all the thioester bonds that join ubiqui- to promote E2-substrate contact. This is consistent with the tin and UbcH5b in the self-assembled UbcH5bUb conjugates report that the E2Ub conjugate, when bound to E3 ubiquitin can potentially be attacked by the 3-amino groups of substrate ligase, is juxtaposed with substrate proteins (Saha and De- lysine residues to form isopeptide bonds. This model accounts shaies, 2008). for previous reports that the UbcH5 family efficiently catalyzes In this study, we determined crystal structure of the multiple ubiquitination coupled with the Cullin-based E3 (Kirkpa- UbcH5bUb intermediate state for ubiquitin transfer and, trick et al., 2006; Rodrigo-Brenni and Morgan, 2007; Saha and furthermore, provided structural insights into the formation of Deshaies, 2008; Windheim et al., 2008). The self-assembled polyubiquitin chains. Not only conformational variability of E3, E2Ub conjugates may transfer several ubiquitins simulta- but also the self-assembling property of the UbcH5bUb conju- neously to the substrate, resulting in multiple ubiquitination. gates with multiple catalytic sites, contribute to catalytic func- Our model also explains why UbcH5 forms a branched polyubi- tions. In conclusion, we suggest that the assembly of E2Ub quitin chain linked through multiple lysine residues of ubiquitin conjugates mediated by the backside interaction is one of the (Kim et al., 2007; Kirkpatrick et al., 2006). Not only substrate molecular strategies employed for efficient and versatile ubiqui- proteins, but also ubiquitin itself, is subjected to multiple transfer tination of substrates.

Figure 6. The Self-Assembled UbcH5bUb Conjugates Enhance the Formation of Polyubiquitin Chain (A) Evaluation of E2 activities of the UbcH5b mutants using in vitro autoubiquitination of MBP-Rma1. (B) Effects of the UbcH5bUb conjugates on the ubiquitination activity of UbcH5b(F62A). Increasing amounts of the UbcH5bUb conjugate with the ester linkage were mixed with MBP-Rma1, E1, ubiquitin, ATP, Mg2+, and UbcH5b or UbcH5b(F62A) at 37C for the indicated times. Reaction products were analyzed by SDS- PAGE, followed by immunoblotting with anti-ubiquitin antibody.

EXPERIMENTAL PROCEDURES ACCESSION NUMBERS

Protein Expression and Puriﬁcation The atomic coordinates of the UbcH5bubiquitin conjugate have been depos- Standard polymerase chain reaction methodology was used to generate ited in the Protein Data Bank with the accession code 3A33.

UbcH5b mutants. Recombinant His6-human UbcH5b, its mutants, and human ubiquitin were produced in Escherichia coli, and His6-E1 (Uba1) was produced SUPPLEMENTAL INFORMATION from baculovirus-infected HiFive insect cells as described previously (Sakata et al., 2007). For NMR measurements, UbcH5b and its mutants were ex- 15 Supplemental Information includes Supplemental Experimental Procedures, pressed in E. coli in M9 minimal medium supplemented with [ N] NH4Cl and 13 three ﬁgures, and one table and can be found with this article online at [ C6] glucose. The samples for two-dimensional NMR experiments were doi:10.1016/j.str.2009.11.007. prepared at labeled-protein concentrations of 0.2 mM and 0.5 mM, respec- 2 2 tively, in 90% H2O/10% H2O (v/v), 50 mM MES buffer, and 1 mM [ H10] dithio- threitol at pH 6.0. ACKNOWLEDGMENTS

Ubiquitin Conjugation Reaction We thank Noriyuki Matsuda, Yukiko Yoshida, and Yasushi Saeki (Tokyo Metro- The UbcH5b (C85S) was charged with ubiquitin by incubating it with 1.0 mM politan Institute of Medical Science, Tokyo, Japan) for useful discussion, and E1 enzyme and a 2-fold molar excess of ubiquitin in 50 mM Tris-HCl (pH Kazuhiro Iwai (Osaka University, Osaka, Japan) for providing the recombinant -1 E1 construct. We thank Kiyomi Senda and Kumiko Hattori for their help in the 7.5), 5 mM MgCl2, 10 mM creatine phosphate, 0.6 U ml creatine phosphoki- nase, 10 mM ATP, and 1 mM DTT overnight at 37C. The resultant preparation of recombinant proteins. This work was supported by Grant-in-Aid UbcH5bUb conjugate was then purified on a RESOURCE S cation exchange for Scientific Research on Priority Area (18076003), Grant-in-Aid for Scientific chromatography column in 20 mM acetate buffer (pH 4.9) and developed with Research on Innovative Areas (20107004), Grant-in-Aid for Scientific Research a 0–1.0 M NaCl gradient. (B) (21370050), and Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology. E. S. and M. Y.-U. are recipients of JSPS Research Fellowships for Young Scientists. Crystallization, Data Collection, Structural Determination, and Refinement Received: June 9, 2009 The purified UbcH5bUb conjugate was concentrated to 1.5 mM and dialyzed Revised: June 26, 2009 in 20 mM Tris-HCl (pH 7.5). Crystals were grown using hanging drop vapor Accepted: November 9, 2009 diffusion at 293 K, mixing equal volumes of the protein conjugate with a well Published: January 12, 2010 solution containing 0.1 M potassium acetate (pH 4.1) and 2.0 M NaCl. For crystal structure analysis, a data set was collected using synchrotron radiation (1.0000 A˚ wavelength) at BL5A of Photon Factory (PF), High Energy Acceler- REFERENCES ator Research Organization (KEK). The diffraction data were processed using HKL2000 (Otwinowski and Minor, 1997). The UbcH5bUb crystal belongs to Baboshina, O.V., and Haas, A.L. (1996). Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by the hexagonal space group P6122 with one UbcH5bUb conjugate per asym- metric unit. The crystal structure of the UbcH5bUb was solved by the molec- 26 S proteasome subunit 5. J. Biol. 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Ubiquitin Intermediate: Insight Into the Formation of the Self-Assembled E2Ub Conjugates

Ubiquitin Intermediate: Insight Into the Formation of the Self-Assembled E2Ub Conjugates