OTUB1 co-opts Lys48-linked ubiquitin recognition to suppressE2 enzyme function

Yu-Chi Juang1,*, Marie-Claude Landry1,*, Mario Sanches1, Vinayak Vittal3, Charles Leung1,Derek F. Ceccarelli1, Abigail-Rachele F. Mateo1,2, Jonathan N. Pruneda3, Dan Mao1, RachelK. Szilard1, Stephen Orlicky1, Meagan Munro1, Peter S. Brzovic3, Rachel E. Klevit3, FrankSicheri1,2,#, and Daniel Durocher1,2,#

1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto,Ontario, M5G 1X5, Canada2Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto,Ontario, M5S 3E1, Canada3Department of Biochemistry, University of Washington, Seattle, WA 98195

SUMMARYUbiquitylation entails the concerted action of E1, E2 and E3 enzymes. We recently reported thatOTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidaseactivity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme forRNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate thestructural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizesubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme.Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1catalytic site and the C-terminus of free ubiquitin, a configuration that mimics the products ofLys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chainrecognition to suppress ubiquitin conjugation and the DNA damage response.

INTRODUCTIONConjugation of ubiquitin (Ub) onto substrates regulates the abundance, localization andactivity of a large fraction of the proteome. Ubiquitylation is a multi-enzymatic process thatfirst necessitates the activation of the terminal carboxyl group of Ub by an E1 enzyme(Pickart, 2001). Activated Ub is transferred to an E2 conjugating enzyme to form a high-

© 2012 Elsevier Inc. All rights reserved.#Address correspondence to: Daniel Durocher, Ph.D., Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 1073, 600University Avenue, Toronto, ON M5G 1X5, CANADA, Tel: 416-586-4800 ext. 2544, durocher@lunenfeld.ca. Frank Sicheri, Ph.D.,Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 1090, 600 University Avenue, Toronto, ON M5G 1X5, CANADA,Tel: 416-586-8471, sicheri@lunenfeld.ca.*These authors contributed equally to the workSUPPLEMENTAL INFORMATIONSupplemental Information includes 7 figures and Supplemental Experimental Procedures and can be found with this article online at:ACCESSION NUMBERSThe structures described in the manuscript have been deposited to the Protein Databank (PDB) with accession codes 4DDI and 4DDG.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Published in final edited form as:Mol Cell. 2012 February 10; 45(3): 384–397. doi:10.1016/j.molcel.2012.01.011.

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energy thioester intermediate, denoted E2~Ub. E2~Ub is bound by an E3 Ub ligase tocatalyze the formation of an isopeptide bond between an amino group, usually the ε-NH2 oflysine, and the C-terminus of Ub. Ubiquitin possesses eight potential acceptor sites andtherefore Ub conjugation can be repeated to form chains. Remarkably, the nature of theubiquitylation products often dictates biological outcome. For example, mono-ubiquitylationof surface receptors is a signal for endosomal sorting (Raiborg and Stenmark, 2009), Lys48(K48)-linked Ub chains target proteins for degradation by the 26S proteasome and Lys63(K63)-linked Ub chains are non-proteolytic signals that nucleate protein-protein interactions(Behrends and Harper, 2011). Given that the complexity and importance of ubiquitylationrivals that of phosphorylation-based signaling networks, ubiquitylation must be highlyregulated.

Ubiquitylation is a reversible post-translational modification. The Ub-substrate isopeptidebond can be cleaved by a group of peptidases called deubiquitylases (DUBs) (Komander etal., 2009). Sequence analysis and functional studies identified 95 potential DUBs encodedby the human genome grouped into five families: ubiquitin C-terminal hydrolases (UCHs),ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), the Josephins andJab1/MPN/Mov34 metalloprotases (JAMM/MPN+). The first three groups are cysteineproteases whereas the latter two are zinc metalloproteases (Komander et al., 2009; Nijman etal., 2005). Of the 90 or so DUBs for which experimental evidence of expression exists, 79are predicted to have deubiquitylase activity (Nijman et al., 2005). The relatively largenumber of putative DUBs without the required infrastructure to support proteolysis suggeststhat some DUBs have evolved protease-independent functions.

The response to DNA double-strand breaks (DSBs) is an example of a cellular pathway thatrelies extensively on protein ubiquitylation (Al-Hakim et al., 2010). Upon DSB detection,the ATM protein kinase initiates a cascade of protein recruitment on chromatin that can bevisualized as discrete nuclear foci when examined by immunofluorescence microscopy(Lukas et al., 2011). The RING-type E3 RNF8 acts downstream of ATM to initiate a Ub-dependent cascade of protein recruitment (Al-Hakim et al., 2010; Huen et al., 2007; Kolas etal., 2007; Mailand et al., 2007). RNF8-dependent ubiquitylation of chromatin elicits therecruitment of a second RING-type E3, RNF168, which elaborates a K63-linked Ub chainon chromatin with its cognate E2, UBC13 (Doil et al., 2009; Stewart et al., 2009). Thisamplification of the ubiquitylation signal triggers the recruitment of additional DNA repairproteins such as RAD18, BRCA1 and 53BP1 to promote DSB repair (Bekker-Jensen et al.,2010; Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Stewart et al., 2009).

In a search for DUBs that oppose the action of RNF168, we identified OTUB1, an OTUfamily member, as a potent suppressor of ubiquitylation at DSB sites (Nakada et al., 2010).Depletion of OTUB1 resulted in a striking persistence of conjugated Ub and 53BP1 focilong after bulk DNA repair was completed (Nakada et al., 2010). This finding was initiallysurprising since OTUB1 is highly selective for K48-linked Ub chains, while RNF168promotes the formation of K63-linked chains on chromatin. This paradox was solved whenOTUB1 was found to act non-catalytically to inhibit the DNA damage response through thebinding of UBC13. OTUB1 binds preferentially to the Ub-charged E2 in a manner thatrequires an N-terminal extension to its core OTU domain (Nakada et al., 2010; Wang et al.,2009). Furthermore, proteomic analyses indicated that OTUB1 binds to E2s of the UBE2Dand UBE2E families including the E2 UbcH5 (Nakada et al., 2010; Sowa et al., 2009).Interestingly, OTUB1 was recently shown to modulate p53 stability through inhibition ofUbcH5 (Sun et al., 2011).

In this study, we sought to elucidate the structural mechanism by which OTUB1 recognizesand inhibits UBC13, UbcH5 and related E2 enzymes. We present a genetically and

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biochemically validated X-ray crystallographic model that explains how OTUB1 recognizesUb-charged E2s. Surprisingly, we found that in addition to binding the E2~Ub, OTUB1 alsobinds free Ub. Remarkably, the E2-linked and free Ub moieties mimic the configuration ofcleaved K48-linked di-Ub chain, the canonical product of OTUB1 deubiquitylase activity.We conclude that OTUB1 co-opts a mechanism akin to product inhibition to inhibit anotherclass of enzymes.

RESULTSA genetic system to dissect E2 inhibition by OTUB1

To understand the mechanism of action of OTUB1 on UBC13, we introduced a plasmidexpressing human OTUB1, tagged with the Flag epitope, in budding yeast under the controlof the GAL1/10 galactose-inducible promoter. Our rationale was to test whether OTUB1 caninhibit yeast Ubc13. To our surprise, since Ubc13 is not essential in yeast, we observed thatinduction of OTUB1 expression severely curtailed yeast growth (Figure 1A). Growthinhibition was independent of the OTUB1 DUB activity, as the C91S mutation, whichrenders OTUB1 catalytically inert, also suppressed growth (Figure 1A). This growthinhibition phenotype was reminiscent of the isopeptidase-independent suppression of E2enzyme action by OTUB1 (Nakada et al., 2010). In support of the possibility that the growthinhibition in yeast was due to E2 inhibition by OTUB1, we found that the OTUB1ΔN

mutant, which is unable to inhibit E2 enzymes, failed to block growth (Figure 1A). Theseresults strongly suggested that OTUB1 expression in yeast shuts down the activity of one ormore E2 enzymes required for growth.

The growth suppression imparted by OTUB1 expression provided a convenient geneticassay for identifying mutations that disable the ability of OTUB1 to inhibit E2 enzymes.Indeed, we readily isolated two strains that were spontaneously resistant to OTUB1expression. Sequence analysis of the recovered OTUB1 plasmids revealed that both strainscontained the same mutation in the OTUB1 open reading frame, P263L (Figure 1B).

To uncover additional mutants, we carried out a random mutagenesis screen (Figure 1C) andscreened over 3 × 107 gap-repair transformants. We identified 104 colonies that wereresistant to OTUB1 expression. We then verified Flag-OTUB1 expression to identify theclones most likely to contain missense mutations. After sequencing, individual mutationswere generated anew by site-directed mutagenesis for confirmation. We ultimately identified7 OTUB1 mutations that allow yeast to growth in spite of OTUB1 expression (Figure 1D).Mutations were scattered across the OTUB1 open reading frame, hinting that multiplesurfaces on OTUB1 are required for its ability to inhibit E2 enzymes (Figure S1).

Characterization of OTUB1 mutants in human cellsWe next sought to determine whether the OTUB1 mutations identified in yeast impacted theresponse to DSBs in human cells. We employed a gene-replacement strategy in whichmutations were introduced in an OTUB1 expression vector that directs expression of anmRNA that is resistant to a potent OTUB1 siRNA (Nakada et al., 2010). These plasmidswere transfected into U2OS cells in which endogenous OTUB1 expression was silenced bysiRNA (Figure S2A). OTUB1 depletion resulted in hyper-activation of RNF168-dependentchromatin ubiquitylation, which can be detected by an increase in conjugated Ub foci at sitesof DNA damage 24 h after 3 Gy of X-irradiation (Figure 2). Reintroduction of wild typeOTUB1 restored normal levels of Ub foci kinetics indicating that the expression levels oftransiently transfected OTUB1 recapitulated endogenous regulation of the DSB response byOTUB1 (Figure 2AB). Interestingly, reintroduction of OTUB1C91S produced a strongerphenotype than wild type OTUB1, which was manifested by a reduced number of IR-

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induced conjugated Ub foci at the 4 h time point. OTUB1C91S was expressed at the samelevel as OTUB1 wild type (Figure 2C) indicating that the C91S mutation results in a proteinwith enhanced potency in inhibiting the DSB response. Lastly, reintroduction of theOTUB1ΔN mutant failed to rescue OTUB1 depletion, as high levels of IR-inducedconjugated Ub foci were detected at 24 h post-irradiation. The OTUB1ΔN mutant proteinwas expressed at the same level as wild type OTUB1 (Figure 2C) ruling out the possibilitythat the failure to rescue OTUB1 depletion was due to varying proteins levels. The activityprofiles of transiently transfected OTUB1 wild type, C91S and ΔN expression vectorsclosely matched those of stably transfected cells that expressed the same proteins at levelsthat approached endogenous OTUB1 levels (Figure S2B). Together, these results indicatedthat the transient reintroduction of siRNA-resistant OTUB1 plasmids in OTUB1-depletedcells largely recapitulated endogenous regulation of the DSB response by OTUB1.

We next tested whether the Q33R, I37T, Q39L, A116T, T134R, D137G, F190S, Y261H andP263L OTUB1 mutants could rescue the OTUB1 depletion phenotype (Q39L was added toour list as a control mutation not predicted to inhibit the DSB response). We introducedthese mutations in the C91S background to provide a greater dynamic range to our assay.We observed complete concordance between the results obtained in yeast and thoseobserved in human cells: mutants that permitted yeast growth also lacked the ability todampen RNF168-dependent chromatin ubiquitylation (for example, I37T and T134R; Figure2). Conversely, mutants that ablated yeast growth also potently ablated the DNA damageresponse (for example, A116T; Figure 2). Each mutant was expressed at the same level asthe wild type and C91S OTUB1 proteins (Figure 2C) ruling out the possibility that theactivity profile was due to varying levels of protein expression. Together, these resultsidentified the Q33, I37, T134, D137, F190, Y261 and P263 residues as being critical for theability of OTUB1 to dampen RNF168-dependent Ub conjugation at DSB sites.

Crystallization of OTUB1 bound to a charged E2To illuminate how the residues mapped above function in the inhibition of Ub conjugation,we initially carried out crystallization screens with a complex containing Ub-chargedUbcH5bC85S (Ub~UbcH5b) and free OTUB1. The C85S variant of UbcH5b was utilizedbecause the oxyester linkage endowed by the serine residue stabilized the Ub-charged state.The trials yielded numerous hits corresponding overwhelmingly to Ub~UbcH5b alone. Toassist identification of crystals containing the desired complex, we employed a UbcH5b-OTUB1 fusion construct (see Supplementary Methods for construct design and rationale).The fusion was subjected to Ub charging reactions with GST-Ub and E1 followed bypurification using glutathione affinity chromatography. Following removal of the GSTmoiety by TEV cleavage, excess free Ub was separated from Ub~UbcH5bC85S-OTUB1 bysize-exclusion chromatography. Crystallization screens with this covalent complex yieldedtwo related monoclinic crystal forms that allowed collection of 3.8 Å (C2) and 3.3 Å (P21)data sets (Table 1).

Structure determination and overview of binding topologyThe C2 crystal contents consist of three biological complexes in the asymmetric unit. Asshown in Figure 3A, each complex, denoted Ub~UbcH5b-OTUB1-Ub, contains onemolecule of UbcH5b covalently linked to Ub, one molecule of OTUB1, and also one freeUb (coloured cyan, pink, green, and yellow, respectively).

The C2 crystal structure was solved by molecular replacement. OTUB1 (PDB 2ZFY)lacking 45 N-terminal residues (Edelmann et al., 2009) and UbcH5b (PDB 2ESK) (Ozkan etal., 2005) were used as search models and were unambiguously positioned in the unit cell inclose contact using Phaser (McCoy et al., 2007). Following iterative rounds of restrained

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refinement, analysis of electron density maps revealed a 22-residue α-helix (denoted helixαA), corresponding to the missing N-terminus of OTUB1, in close contact with the E2-linked donor Ub. Inclusion of the donor Ub and the OTUB1 helix αA into model refinementrevealed unexpected electron density for a free Ub molecule engaging the distal Ub bindingsite of OTUB1 (Messick et al., 2008). A near-complete model of the Ub~UbcH5b-OTUB1-Ub complex was refined at 3.8 Å resolution to an Rfactor/Rfree of 21.7/27.9 % with goodgeometry statistics (Table 1). Resolution was extended by refinement at 3.3 Å resolutionagainst the P21 X-ray data set to an Rfactor/Rfree of 22.1/28.8%, with a doubling of thecontents of the asymmetric unit

The fold architectures of UbcH5b, OTUB1 and Ub have been described previously(Edelmann et al., 2009; Ozkan et al., 2005; Vijay-Kumar et al., 1987) and do not deviatesignificantly in the Ub~UbcH5b-OTUB1-Ub complex (see Figure S1 for sequencealignments and secondary structure assignments). A detailed description of the binaryinteractions defining the overall topology of the Ub~UbcH5b-OTUB1-Ub complex follows.

Binding interface between OTUB1 and UbcH5bUbcH5b engages two non-contiguous surfaces on OTUB1 (Figures 3AB and S3A) burying atotal surface area of 2001 Å2. The larger surface on OTUB1 is centered on helices α5, α7,and the α9-α10 linker, which engages a surface composed of helix α1, the α2-α3 linker, andthe β3-β4 linker on UbcH5b. Specific side-chains that mediate hydrophobic and charge-complementary interactions include Phe130, Thr131, Phe133, Thr134, Ile135, Asp137,Phe138, Asp169, Tyr170, Val173, Leu177, Gln206, Glu209, Pro210, Met211, and Lys213on OTUB1 and Met 1, Arg5, Lys8, Asp12, Lys101, Pro61, Phe62, K63, Gln92, Ser94, Pro95, Ala96, Thr98 and Lys101 on UbcH5b.

A smaller contact surface on OTUB1 is centered on the helix αA, which engages thecatalytic cleft region of UbcH5b in a manner that partly shields the labile thioester (in thiscase oxyester) linkage to donor Ub (Figures 3AB and S3A). Specific residues that mediatethis contact include Asp27, Glu28, Met31 and Asp35 on OTUB1 and K63, Lys66, Ser83,Arg90, and Ser91 on UbcH5b.

The surface engaged by OTUB1 overlaps with the E3-binding surface on UbcH5b (FigureS4AB). Occlusion of the E3-binding surface, along with the shielding of the Ub~UbcH5bester linkage by OTUB1 likely accounts for the ability of OTUB1 to suppress E2 function.The surface of UbcH5b engaged by OTUB1 is relatively well conserved across the two E2enzyme subfamilies to which OTUB1 was previously shown to bind (Figure S1). Thus wepredict that the binding mode uncovered between OTUB1 and UbcH5b is of generalrelevance to all E2s targeted by OTUB1.

To verify that the interactions observed in the crystal structure are also observed in solution,the binding of the E2s UbcH5c (closely related to UbcH5b and also targeted by OTUB1)and UBC13 to full-length OTUB1 was examined by NMR spectroscopy. A series of(1H, 15N)-HSQC spectra of UbcH5c~Ub in which the E2 subunit was 15N-labeled wasmixed with increasing concentrations of OTUB1 (Figure S5). Chemical shift perturbationsof backbone amide NMR resonances are sensitive indicators of their environment and can beused to map protein-interaction surfaces. Resonances corresponding to residues in α helix 1,in loops 4 and 7, and the UbcH5c active site (N77 and L86) are perturbed upon addition ofOTUB1 (Figure 3C). The UbcH5 surface defined by the observed NMR resonanceperturbations is the same as that observed in the crystal structure (Figure 3D). The presenceof a conjugated Ub subunit does not appreciably alter E2 residues involved in bindingOTUB1. NMR mapping experiments with both 15N-UbcH5c~Ub and free 15N-UbcH5cdefined the same binding surface. Corresponding NMR experiments with15N-UBC13

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showed that the same E2 structural elements are also used by UBC13 to bind OTUB1(Figure 3C). These results strongly suggest that the closely related E2s that interact withOTUB1 do so in a similar manner. In contrast, UbcH7, which is not inhibited by OTUB1,does not interact with OTUB1 in our NMR experiments (Figure S5B).

When we model UBC13 binding to OTUB1, the surface of UBC13 engaged by its ancillaryfactor UEV2 (Mms2) (Eddins et al., 2006) is not occluded significantly by OTUB1 (FigureS4C). This is consistent with the observation that binding of UBC13 to UEV1A or OTUB1is not mutually exclusive (Nakada et al., 2010). While the binding site on UbcH5b for E1also overlaps with OTUB1 (Figure S4D), the strong binding preference of OTUB1 for theUb-charged form of E2s could explain why OTUB1 does not inhibit charging of targetedE2s by E1. The binding surface of OTUB1 on UbcH5b does not overlap with either thepreviously characterized binding surface for non-covalently bound Ub (Brzovic et al., 2006)or with the predicted binding surface for donor Ub (Hamilton et al., 2001; Wickliffe et al.,2011) (Figure S4EF). Thus, direct occlusion of Ub-binding surfaces on the E2 is unlikely tocontribute to OTUB1 E2 inhibitory function.

Binding interface between donor Ub and OTUB1With the exception of the covalent tether between the C-terminus (Gly76) of the donor Uband the catalytic cysteine (in this case Ser85) of UbcH5b, the donor Ub makes little contactwith UbcH5b (total buried surface area of contact = 262Å2). Since the association of donorUb with the donor Ub binding surface of E2s has been shown previously to be important forthe Ub transfer function of some E2s (Saha et al., 2011; Wickliffe et al., 2011), thisstructural feature may also contribute to the E2 inhibitory function of OTUB1. Instead, thedonor Ub engages two surfaces on OTUB1 (Figures 3A, 4A and S3B) burying a totalsurface area of 1700 Å2. The first contact surface involves loop regions (α-α2, β2-α3, β4-β5)on the core domain of OTUB1. Specific contact residues involved on OTUB1 includeTyr61, Ala62, Asp65, Tyr68, Pro87, Asp237, Pro263, and His265. The reciprocal contactsurface on the Ub moiety is comprised by a segment spanning strands β3 to β5. Specificcontact residues involved on Ub include Lys48, Arg54, Ser57, Asp58, Tyr59, Asn60 andGln62. A second contact surface involves the N-terminal helix αA of OTUB1, whichengages the β-sheet surface (β1, β3, β4 and intervening linkers) of Ub. Specific Ub-contacting residues on OTUB1 include Tyr26, Ile30, Gln33, Gln34, Arg36, Ile37, Gln38,and Ile41. Specific contact residues on the donor Ub include Lys6, Leu8, Arg42, Ile44,Gly47, His68, Val70, Leu71, and Leu73.

The interaction of donor Ub with the N-terminal helix of OTUB1 is strikingly reminiscent ofthe interaction between Ub interaction motifs (UIMs) and Ub (Fisher et al., 2003). Both theOTUB1 αA- and UIM interactions involve the Ile44 hydrophobic patch of Ub engaging asingle α-helix (Figure 4B). Small differences in binding mode include the interaction angle,which differs by ~23°, and the closest distance of approach, which is smaller in the Ub-UIMinteraction by ~4 Å due to bulkier residues on the OTUB1 helix (for example Ile37) ascompared to the corresponding residue (Ala) in canonical UIMs. In support of the Ile44hydrophobic patch mediating important contact with OTUB1, UBC13 charged with an I44AUb mutant (UBC13~UbI44A) was compromised for its ability to bind to OTUB1 (Figure4C).

Binding interface between distal Ub and OTUB1Perhaps the most surprising feature of the structure was the presence of a non-covalentlylinked Ub molecule bound to OTUB1 (Figures 3A, 5A, S3C). The free Ub moiety interactswith OTUB1 at its distal Ub-binding site in a manner nearly identical to yeast Otu1covalently bound to Ub-Br3 (Messick et al., 2008) (Figure S6A) burying a total surface area

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of 2344 Å2. The interaction surface on the distally-bound Ub consists of the solvent exposedβ-sheet (β1, β3, β4) and flanking loop regions which engage an extensive concave pocket onOTUB1 comprised of helix α10, strands β3, β3′, β4, and an irregular loop region connectinghelices α7 and α9. Contact residues on OTUB1 include Phe189, His192, Phe193, Glu195,Glu214, Ser215, Asp216, His217, Ile218, Ile221, Gln225, Tyr235, Asp237, Asn245,His247, Phe249, Glu251, Tyr261, and Tyr266. Contact residues on Ub include Leu8, Thr9,Ile36, Pro37, Asp39, Gln40, Arg42, Ile44, Gly47, His68, Val70, Leu71, Leu73, Gly75 andGly76.

While the presence of free Ub in the OTUB1 complex was unexpected, reanalysis of ourpurification scheme revealed consistent levels of free Ub in our Ub~UbcH5b-OTUB1preparations. Interestingly, the C-terminal tail of free Ub straddles the catalytic cleft ofOTUB1 providing part of the platform engaged by the donor Ub (total buried surface area ofcontact = 378Å2). This affords van der Waals and salt bridge (between Glu51 in donor Uband Arg72 and Arg74 in free Ub) interactions between Ub molecules (Figure 5B), which wesuspected could confer interdependency of Ub~UbcH5b and Ub binding to OTUB1.

Molecular recognition of K48-linked di-UbThe isopeptidase activity of OTUB1 is notable for its exquisite specificity for K48-linkedUb chains (Edelmann et al., 2009). In this context, the relative position of the two Ubmolecules engaged by OTUB1 that positions K48 of the donor Ub in immediate proximityto the C-terminus of the distally bound Ub was remarkable (Figure 5BC). We reasoned thatthis binding configuration might reflect how OTUB1 recognizes its preferred substrate,K48-linked Ub chains. Indeed, when we model a K48 linkage between the Ub molecules,the structure is easily accommodated without changes to main chain conformation (Figure5C). In addition, the active site cysteine is well positioned to attack the K48-linkedisopeptide bond. Interestingly, the side chain of His265 in the OTUB1 active site hydrogenbonds to the side chain of Cys91, a characteristic feature of all papain-like protease activesites in their productive conformations. This is in contrast to the apo-OTUB1 structure, andsuggests that the binding of the OTUB1 substrate may induce a productive conformation ofthe active site.

Mapping of OTUB1 mutations onto the Ub~E2-OTUB1-Ub structureWe next mapped the position of OTUB1 mutations identified in our genetic screen onto thestructure of the Ub~E2-OTUB1-Ub complex. Each mutation identified was located on oneof four distinct inter-subunit interaction surfaces, lending strong support to the functionalrelevance of our structural model (Figure 6A). Firstly, the I37T and Q33R mutations map tothe OTUB1 helix αA-donor Ub interface (Figure 4A), indicating that at the genetic level, theOTUB1 UIM-like-donor Ub interaction is critical for the formation or function of theinhibitory complex. Consistent with this model, I37T and Q33R, but not Q39L (which doesnot inhibit the DNA damage response), inhibited di-Ub synthesis by UBC13 (Figure 6B).Secondly, the T134R and D137G mutations map to the E2-OTUB1 interface (Figure 3B),with the T134R mutation predicted to have a more drastic impact on the E2 interaction byvirtue of insurmountable steric clashes. In support of this prediction, OTUB1T134R is fullycompromised in its ability to inhibit UBC13-dependent di-Ub synthesis (Figure 6B).OTUB1D137G also failed to inhibit UBC13 (Figure 6B). Thirdly, the Y261H and P263Lmutations map to the OTUB1 core domain-donor Ub interface (Figure 4A) furthersupporting the importance of OTUB1-donor Ub contacts for recognition and inhibition ofthe charged E2. Accordingly, P263L failed to inhibit Ub conjugation by UBC13 (Figure6B). Fourthly, the F190S mutation maps to the OTUB1-free Ub interface where, inconjunction with Phe189, Phe193 and Ile221, it comprises an elaborate hydrophobic pocketthat engages Leu8 of Ub (Figure S6B). Consistent with a functional importance of this

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pocket, the F190S mutation disrupted the ability of OTUB1 to suppress UBC13 activity invitro (Figure 6B).

To further probe the importance of free Ub binding to OTUB1, we designed an E195Rmutation, which we predicted to potently impair binding to free Ub. When introduced intoyeast, OTUB1E195R was unable to suppress yeast growth (Figure 6C) and when introducedin human cells, it failed to rescue conjugated Ub foci clearance caused by OTUB1 depletion(Figure S7A). These results further validate the functional importance of Ub engagement tothe distal Ub-binding site on OTUB1. Finally, we carried out GST pull-down assays withthe OTUB1 mutants that failed to inhibit UBC13 and found that all had reduced binding toUb~UbcH5b and free Ub (Figure S7B).

Since the E2-linked donor Ub and distally bound free Ub occupy the inferred substraterecognition sites for K48 chain hydrolysis, we investigated the impact of OTUB1 mutationson its deubiquitylase activity. We employed a FRET-based assay with a substrate consistingof a K48-linked di-Ub with an internally quenched fluorophore. Cleavage results in ahomogeneous fluorescence signal that can be followed in a time-resolved manner. In thisassay, OTUB1 shows robust time-dependent activity whereas OTUB1C91S is completelyinactive (Figure 6DE). We first assayed OTUB1 mutations at the OTUB1-E2 interface(T134R and D137G). Both mutants displayed robust DUB activity (Figure 6D). Next, weexamined the mutations that affect residues located at the various OTUB1-Ub interfaces(I37T, Q33R, P263L, F190S and E195R). In contrast to mutations at the OTUB1-E2interface, mutations at each of the OTUB1-Ub interfaces greatly impaired OTUB1deubiquitylase activity (Figure 6E). Together, the inhibitory profile of the OTUB1 mutantswas fully consistent with the crystal structure of the Ub~E2-OTUB1-Ub complex anddemonstrated (1) that the E2 interaction surface on OTUB1, remote from its catalytic site,was dispensable for its DUB function, and (2) that both Ub interaction surfaces on OTUB1were required for its isopeptidase activity, as they likely engage K48-linked di-Ubsubstrates. Furthermore, the results identified the T134R mutation as a true separation-of-function mutation, as it impaired E2 inhibitory function without impairing the DUB activityof OTUB1.

The strong impact of the I37T and Q33R mutations on deubiquitylase activity was surprisingsince OTUB1ΔN retained some level of catalytic activity against unlabeled di- or tetra-Ubsubstrates (Edelmann et al., 2009; Nakada et al., 2010). To probe this conundrum further, weassessed how OTUB1ΔN behaved in our FRET-based DUB assay. As shown in Figure 6E,OTUB1ΔN was largely inactive as a DUB, suggesting that the FRET-based assay wassensitized for perturbations in Ub-binding at the proximal site, possibly due to the presenceof fluorescent and quencher probes on the substrate. While our use of a sensitized substrateto uncover perturbations in Ub-binding was serendipitous, these results are entirelyconsistent with the architecture of the Ub~E2-OTUB1-Ub model obtained bycrystallography.

Insights into the regulation of the OTUB1-E2 interactionHow is the OTUB1-E2~Ub inhibitory complex regulated to release the E2 conjugatingenzyme in its active form, for example during the DNA damage response, remains an openquestion. Our mutagenesis results and the structure of the Ub~E2-OTUB1-Ub complexhowever, offers two possible avenues for consideration. One avenue might entail disruptionof the inhibitory complex by phosphorylation of OTUB1 on one or more of the nineconserved phosphorylatable residues located at essential interaction surfaces. Indeed, ourgenetic screen identified the T134R mutation, which targets one such residue in OTUB1 onits E2-binding surface. To extend this analysis, we mutated Thr134 to a phosphomimeticresidue (T134E) or to a non-phosphorylatable alanine residue (T134A) and assayed the

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ability of OTUB1 to inhibit yeast growth and to rescue the OTUB1 depletion phenotype (inboth cases, the mutations were introduced in the C91S background). Consistent with apossible phosphoregulatory role for Thr134, the non-phosphorylatable mutant OTUB1T134A

inhibited yeast growth (Figure 7A and Figure S7C) and was as potent as the C91S mutant inrescuing OTUB1 depletion in human cells (Figure 7B). In contrast, OTUB1T134E was notfunctional in inhibiting either yeast growth or in restoring the normal kinetics of conjugatedUb foci in OTUB1-depleted cells (Figure 7AB). Furthermore, the OTUB1T134E mutantfailed to inhibit di-Ub synthesis by UBC13 in vitro (Figure 7C). These results demonstratethat phosphorylation could, in principle, regulate the E2 inhibitory function of OTUB1.

A second possible avenue for regulation centres on the role of the distally bound free Ub inthe formation of the OTUB1-E2~Ub complex. Based on the behaviour of the F190S andE195R mutants, which demonstrated an essential role for the distal Ub binding site inOTUB1 E2 inhibitory function, we reasoned that the cellular pool of free Ub could modulateinhibitory complex formation. To assess if free Ub could influence binding of OTUB1 toUbcH5b~Ub, we employed a time resolved (TR)-FRET-based assay where UbcH5b~Ubwas labelled with Tb3+ cryptate and OTUB1 was labelled with Alexa 488. In the absence offree Ub, only weak non-saturating binding between OTUB1 and UbcH5b~Ub was observed.In contrast, complex formation was clearly stimulated in the presence of 1 or 10 μM free Ub(Figure 7D). Further demonstrating an interdependency of binding, we found that free Ubbound to OTUB1 only in the presence of UbcH5b~Ub (Figure 7E). In sum, these datasupport a potential role for free Ub or an alternate form of Ub, in the regulation of OTUB1’sE2 inhibitory function.

DISCUSSIONDespite the fundamental influence of ubiquitylation on most cellular processes, there is apaucity of mechanisms that have been reported to modulate the activity of E2 conjugatingenzymes. One such mechanism is the regulation of a subgroup of E2s that includes UBC13and UbcH5 by OTUB1, a DUB. We found that OTUB1 assembles a complex that placestwo Ub moieties in a configuration that is reminiscent of a cleaved K48 linked di-Ub chain.Our validation studies strongly support the hypothesis that this complex leads to suppressionof E2 enzyme activity by blocking access to the E3, by hindering attack on the E2~Ubthioester bond, and possibly by hindering association of donor Ub with the donor Ub-binding surface of the E2.

Inhibition of an enzyme by its product, a phenomenon termed “product inhibition” inenzymology, has been recognized for over a century (Frieden and Walter, 1963). Productinhibition often provides negative feedback inhibition and has been manipulated by natureand chemists to produce selective enzyme inhibitors. In the case of the OTUB1-E2~Ubinteraction, we have an unusual example of product inhibition mimicry; the assembly of theUb~E2-OTUB1-Ub complex completes the formation of a pseudo DUB cleavage productthat inhibits a second enzyme, in this case the E2.

Since the unique N-terminal extension of OTUB1 is a critical determinant of E2~Ubrecognition, this explains why OTUB2, an OTUB1 paralog that lacks an analogousextension, cannot inhibit E2s. As the N-terminal region of OTUB1 also contributes to K48-linked Ub chain recognition, the lack of this extension in OTUB2 would also explain itsweaker ability to discriminate between Ub chain linkages (Edelmann et al., 2009).Interestingly, TRABID, an OTU family member, appears to employ a similar strategy forK29 and K33 Ub-linkage recognition, whereby an ankyrin repeat domain serves ananalogous function to helix αA in OTUB1 (Licchesi et al., 2011).

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The mechanistic themes underlying K48-linked chain selection by OTUB1 are generallysimilar to those underlying K63-linked Ub chain selection by AMSH-LP (Sato et al., 2009)even though the underlying catalytic domain structures of AMSH-LP and OTUB1 areunrelated. Firstly, both DUBs recognize an extended conformation of a di-Ub unitcharacterized by few inter-Ub moiety contacts (Figure S6C). Secondly, both DUBs makeextensive contacts to both proximal and distal Ub moieties (1,324 Å2 and 472Å2,respectively in the case of AMSH-LP). The former point is somewhat surprising since K48-linked Ub chains, unlike K63-linked chains, adopt a more compact topology involvingextensive inter Ub moiety contacts (Varadan et al., 2004). While K48-linked Ub chains maybe more compact in solution, this feature might hinder accessibility to the scissile isopeptidebond. We posit that the need for accessibility to the scissile bond will impose a generalrequirement for an extended conformation of Ub chains irrespective of the preferred isolatedchain conformation in solution.

One corollary of our model is that concentration of OTUB1 should be comparable to that ofthe Ub-charged E2s it inhibits. Indeed, we found that OTUB1 is more abundant than UBC13in U2OS and 293T cells (Figure S7D). If one considers that OTUB1 binds primarily to theUb-charged form of E2s, these OTUB1 levels are sufficient to regulate UBC13 action andthe DSB response. Upon induction of the DSB response, the Ub~UBC13-OTUB1interaction must be relieved to enable UBC13 to act with RNF168. Our mutagenesis andstructural studies provide a few clues that will be the focus of future study. One avenue ofregulation involves potential post-translational modification of OTUB1 at inter-subunitcontact surfaces. While our data are consistent with a potential phosphoregulatory role atThr134, phosphorylation at this site has yet to be detected experimentally. A second possibleavenue of regulation involves engagement by OTUB1 of Ub at the distal Ub-binding site. Itis conceivable that local or compartmentalized fluctuations of Ub levels might be achievedby bursts of DUB activity or, conversely, E3 ligase activity. In support of this possibility,FRAP experiments examining chromatin ubiquitylation at UV lesions indicated that Ublevels are highly dynamic at sites of DNA damage. (Marteijn et al., 2009).

EXPERIMENTAL PROCEDURESYeast strains and plasmids

Standard yeast methods were used for growth and genetic modification. All strains derivefrom BY4741. The OTUB1 plasmids used in this study were constructed by introducing aHindIII-XhoI fragment from pcDNA3-Flag-OTUB1 (Nakada et al., 2010) into pRS425 orpcDNA5/FRT/TO. Site-directed mutagenesis was carried using Quikchange (Agilent).

Protein expression and purificationUbcH5bC85S, UbcH5bC85S-OTUBΔ1-24 and the OTUB1 proteins were expressed inEscherichia coli as TEV-cleavable GST fusions and purified by glutathione affinity, TEVcleavage, and size exclusion chromatography. The Ub-charged versions of UbcH5bC85S andUbcH5bC85S-OTUBΔ1-24 were generated by incubating proteins with 0.5 μM E1 and a 3-fold molar excess of His6-GST-TEV-Ub in 50 mM Tris-Cl (pH 8.5), 150 mM NaCl, 10 mMMgCl2, 5 mM ATP and 1 mM DTT at 37°C overnight. The Ub-charged proteins werepurified from uncharged proteins by glutathione affinity, TEV cleavage, size exclusionchromatography and concentrated to 5 mg/ml in buffer containing 20 mM HEPES (pH7.5),150 mM NaCl.

Deubiquitylase assaysDUB assays were carried out with 100 nM of OTUB1 and 250 nM of the di-Ub (K48) IQFsubstrate #5 (Life Sensors) in a buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 0.3

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% Brij-35, 1 mM DTT, 0.1% BSA. TAMRA fluorescence was read on an Enspire 2300plate reader (Perkin Elmer).

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThe order of MCL and YCJ in the author list was determined by sortition. We thank staff at the Canadian LightSource and I. Kourinov and staff at Argonne National Laboratory at the Advanced Photon Source on theNortheastern Collaborative Access Team beamlines for assistance with diffraction experiments. MCL holds a post-doctoral Fellowship of the Canadian Breast Cancer Foundation. FS is a Canada Research Chair (Tier 1) inStructural Principles of Signal Transduction and DD is the Thomas Kierans Chair in Mechanisms of CancerDevelopment and a Canada Research Chair (Tier 1) in Molecular Genetics of the DNA Damage Response. Thiswork was supported by CIHR grants MOP84297 to DD and MOP57795 to FS, and NIH grant R01 GM088055 toREK.

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Figure 1. A genetic system to dissect E2 inhibition by OTUB1 (see also Figure S1)(A) Left panel: Five-fold serial dilutions of BY4741 strains transformed with the indicatedpRS425 derivatives were spotted onto selective media containing either glucose (uninducedcondition) or galactose (induced) as the carbon source. Right panel: Whole cell extracts(WCE) of the strains shown in the left panel were separated by SDS-PAGE and probed forFlag-OTUB1 expression. Pgk1 immunoblotting was used as a loading control.(B) Left panel: Examination of yeast growth assay as in (A). Right panel: WCE of thestrains shown in the left panel were separated by SDS-PAGE and probed for Flag-OTUB1expression. Pgk1 immunoblotting was used as a loading control.(C) Schematic of the genetic screen used to identify human OTUB1 mutants that fail toinhibit yeast growth.(D) Examination of yeast growth as in (A).(E) Whole cell extracts of the strains shown in (D) were separated by SDS-PAGE andprobed for Flag-OTUB1 expression. Pgk1 immunoblotting was used as a loading control.

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Figure 2. Regulation of RNF168-dependent ubiquitylation by OTUB1 (see also Figure S2)(A–C) U2OS cells were first transfected with non-targeting (CTRL) or OTUB1 siRNAs andthen transfected with the indicated pCDNA5-Flag derivatives. All OTUB1 plasmids weresiRNA-resistant. Cells were then irradiated with X-rays (3 Gy) and processed for Flag orconjugated Ub (FK2) immunofluorescence 0, 4 and 24 h post-IR. DNA was counterstainedwith DAPI. Quantitation of the immunofluorescence data is shown in (A). Data is presentedat the mean +/− S.D. n=3. The dashed line indicates the percentage of control-transfectedcells with FK2 foci 24 h post-irradiation. Representative micrographs ofimmunofluorescence data 24 h post-irradiation are shown in (B). Scale bar=10 μm. Analysisof Flag-OTUB1 expression is shown in (C). Tubulin was used a loading control.

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Figure 3. Structure of the Ub~UbcH5b-OTUB1-Ub complex (see also Figures S1, S3, S4, S5)(A) Schematic and ribbons representation of the Ub~UbcH5b-OTUB1-Ub complex withsubunits coloured pink, cyan, green, and yellow, respectively. The side chains of thecatalytic cysteine of OTUB1 and UbcH5 are shown as sticks. Helix αA of OTUB1 and theN- and C-termini of subunits are labeled where visible.(B) Stereo view of the OTUB1-UbcH5b binding interface. Main chains are coloured as in(A). Contact residues are shown as sticks with carbon atoms coloured according to theirrespective main chain and oxygen, nitrogen, and sulfur atoms are coloured red, blue, andyellow, respectively. The positions of OTUB1 mutants tested in this study are labeled red.(C) UbcH5c and UBC13 utilize similar surfaces to bind OTUB1. Significant perturbations(>1 S.D. from the mean) arising from OTUB1 binding to 15N-UbcH5c (top) or 15N-UBC13(bottom) are mapped in red onto the respective E2 structures.(D) NMR analysis shows binding of OTUB1 to UbcH5c in solution is consistent with X-raydata. Resonances in 15N-UbcH5c significantly affected by binding of OTUB1 are mapped(shown in red) onto the corresponding residues in the OTUB1:UbcH5b~Ub crystal structure.

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Figure 4. Binding mode of donor Ub to OTUB1 (see also Figure S1 and S3)(A) Stereo view of the donor Ub-OTUB1 binding interface. Colouring as in Figure 3B. Thepositions of OTUB1 mutants tested in this study are labeled in red.(B) Stereo comparison of Ub-UIM and donor Ub-OTUB1 helix αA binding modes. TheUIM-Ub structures corresponding to VPS27 (PDBID 1Q0W) and Hrs (PDBID 2D3G) arecoloured grey and orange respectively. The OTUB1-donor Ub structure is coloured as inFigure 3B. In all three complexes, Ub engages its target α-helices using a common surfacecentered on Ile44 (labeled). The N- and C-termini of the UIMs and OTUB1 α-helices arealso labeled.(C) A mixture of uncharged and Ub-charged UBC13C87S that was either prepared with wildtype Ub (UBC13~Ub) or UbI44A (UBC13~UbI44A) were incubated with either GST-OTUB1C91S or, as control, GST-Pcc1. Proteins were separated by SDS-PAGE and stainedwith Coomassie Brilliant Blue.

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Figure 5. Binding mode of free Ub to OTUB1 (see also Figure S1, S3 and S6)(A) Stereo view of the free Ub-OTUB1 binding interface. Coloring is as in Figure 3B. Thepositions of OTUB1 mutants tested in this study are labeled in red. The side chain of Phe190in OTUB1, immediately adjacent to Phe189 and Phe193, is not shown for sake of clarity.For the Phe190 position see Figure S5B.(B) Stereo view of donor Ub and free Ub in the proximity of the catalytic cleft of OTUB1.Coloring is as in Figure 3B. Highlighted as sticks are the catalytic residues of OTUB1(Cys91, His265 and Pro87), and residues in direct contact between donor and free Ub.(C) Stereo view of donor Ub and free Ub in proximity of the catalytic cleft of OTUB1.Coloring is as in Figure 3B. Highlighted as sticks are the catalytic residues of OTUB1(Cys91, His265 and Pro87), K48 of donor Ub and Gly76 of free Ub. Superimposed on thesolved structure is a model of an isopeptide linkage between the K48 side chain of donor Uband the Gly76 C-terminus of free Ub. A productive conformation of OTUB1 catalyticresidues in the Ub~UbcH5b-OTUB1-Ub complex contrasts with a non-productiveconformation observed in the apo-OTUB1 complex (PDB 2ZFY).

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Figure 6. The OTUB1 mutants map to functionally important intermolecular interfaces(A) Position of the OTUB1 mutations identified in the yeast genetic screen on the structure-based model of the Ub~UBC13-OTUB1-Ub complex.(B) Single-turnover ubiquitylation reactions were assembled by pre-incubating UBC13, E1,biotinylated Ub and ATP for 5 min with or without the indicated OTUB1 proteins. To thereaction mixtures were added excess unlabeled Ub, UEV1 and EDTA. The reactions wereincubated for an additional 15 min, separated by SDS-PAGE, transferred onto nitrocelluloseand stained with Ponceau S followed by streptavidin (SA)-HRP. Asterisk denotes a DTT-resistant ubiquitylated UBC13 species.(C) Examination of yeast growth and protein expression as in Figure 1A.(D–E) Graph of internally quenched K48-linked di-Ub cleavage reactions with the indicatedOTUB1 proteins.

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Figure 7. Potential modulation of the OTUB1-E2 interaction(A) Examination of yeast growth as in Figure 1A.(B) Left panel: U2OS cells were first transfected with non-targeting (CTRL) or OTUB1siRNAs and then transfected with the indicated pCDNA3-Flag derivatives and processed asin Figure 2. Quantitation of the immunofluorescence data is shown. Data is presented at themean +/− S.D. n=3. Right panel: examination of Flag-OTUB1 expression. Tubulin was usedas loading control(C) Analysis of di-Ub synthesis by UBC13 in the absence (−) or presence of the indicatedOTUB1 proteins exactly as Figure 6B.(D) Titration analysis of OTUB1 binding to UbcH5b~Ub in the absence or presence of 1 or10 μM free Ub using a time TR-FRET assay. The data presented as the mean +/− S.E.M.n=2.(E) Pull down analysis of GST-OTUB1 binding to free Ub in the presence of 0, 5, 10 and 20μM concentrations of UbcH5b~Ub. Proteins were separated by SDS-PAGE and stained withCoomassie Brilliant Blue.

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Table 1

Data collection and refinement statistics

Dataset 1 Dataset 2

Source APS CLS

Space Group C 1 2 1 P 1 21 1

Unit Cell Parameters

a, b, c (Å) 173.74, 106.23, 134.74 134.57, 104.93, 148.48

α, β,γ (°) 90.00, 123.74, 90.00 90.00, 104.20, 90.00

Wavelength (Å) 0.97922 0.97949

Resolution (Å) 50.0 – 3.80 (4.02- 3.80) 49.29 – 3.30 (3.50 – 3.30)

Completeness (%) 96.7 (83.1) 99.7 (99.4)

Redundancy 3.56 (3.3) 4.25 (4.25)

I/σ(I) 9.44 (1.86) 9.62 (1.46)

Rmeas (%) 16.5 (86.6) 12.6 (121.6)

Refinement Statistics

Rfree/Rfactor 21.7/27.9 22.17/28.79

RMSD Bond Length (Å) 0.006 0.018

RMSD Bond Angle (°) 1.21 1.833

Average B-factor (Å2) 119.98 138.6

Number of protein atoms 9619 26574

Ramachandran

% preferred 92.5 89.3

% allowed 7.5 8.9

% outliers 0 1.8

Values in parentheses are for highest-resolution shell.

Mol Cell. Author manuscript; available in PMC 2013 February 10.