Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

David M. Duda,1,2,4 Laura A. Borg,2,4 Daniel C. Scott,1,2,4 Harold W. Hunt,2 Michal Hammel,3 and Brenda A. Schulman1,2,* 1Howard Hughes Medical Institute 2Departments of Structural Biology and Genetics/Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA 3Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA 4These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.cell.2008.07.022

SUMMARY Knipscheer and Sixma, 2007). In humans, UBL ligation is catalyzed by 600 potential E3s (Li et al., 2008). E3 activity is Cullin-RING ligases (CRLs) comprise the largest generally regulated by posttranslational modifications such as ubiquitin E3 subclass, in which a central cullin phosphorylation, hydroxylation, processing, or UBL attachment. subunit links a substrate-binding adaptor with an The largest E3 subclass consists of modular, multisubunit E2-binding RING. Covalent attachment of the ubiqui- Cullin-RING ligases (CRLs) (Cardozo and Pagano, 2004; Petroski tin-like protein NEDD8 to a conserved C-terminal and Deshaies, 2005). Human CRL cores contain a cullin (Cul1, 2, domain (ctd) lysine stimulates CRL ubiquitination ac- 3, 4A, 4B, 5) and Rbx/Roc1 or 2 and adopt an elongated archi- tecture with substrate- and E2-binding sites at opposite ends tivity and prevents binding of the inhibitor CAND1. (Zheng et al., 2002b). At one end, a cullin’s N-terminal domain Here we report striking conformational rearrange- ctd (ntd) can pair with up to hundreds of substrate-binding adaptors. ments in the crystal structure of NEDD8Cul5 - Distally, the C-terminal domain (ctd) is assembled from 4-helix ctd Rbx1 and SAXS analysis of NEDD8Cul1 -Rbx1 bundle (4HB), a/b, and winged-helix B (WHB) subdomains. The relative to their unmodified counterparts. In NEDD8y- 4HB interacts with the ntd. The a/b subdomain binds Rbx1’s lated CRL structures, the cullin WHB and Rbx1 RING N-strand. Rbx1’s RING, which is thought to bind E2s in the subdomains are dramatically reoriented, eliminating same manner as other E3 RINGs (Zheng et al., 2000), is con- a CAND1-binding site and imparting multiple poten- nected to Rbx1’s N-strand via a 6-residue linker. In unmodified tial catalytic geometries to an associated E2. CRLs, Rbx1’s RING contacts a cullin’s WHB (Zheng et al., Biochemical analyses indicate that the structural 2002b). malleability is important for both CRL NEDD8ylation CRL activity is regulated by both inhibition and activation. Cul1, 2, 3, 4A, and 5 bind CAND1, the CRL assembly inhibitor and subsequent ubiquitination activities. Thus, our (Liu et al., 2002; Zheng et al., 2002a). A Cul1-Rbx1-CAND1 struc- results point to a conformational control of CRL ture showed the two CRL ends as key CAND1-binding sites activity, with ligation of NEDD8 shifting equilibria to (Goldenberg et al., 2004). Subtle differences in the relative loca- disfavor inactive CAND1-bound closed architec- tions of Cul1’s N and C termini observed upon CAND1 binding tures, and favor dynamic, open forms that promote indicate potential for altered orientations of a CRL’s ntd and polyubiquitination. ctd (Goldenberg et al., 2004). Cul1, 2, 3, 4A, 4B, and 5 are activated by ligation of the UBL, NEDD8, to a conserved Lys in the WHB (Figure S1 available on- INTRODUCTION line) (Jones et al., 2008; Pan et al., 2004; Xirodimas et al., 2008). As with ubiquitination cascades, NEDD8’s E2 (Ubc12) binds Covalent attachment of ubiquitin-like proteins (UBLs) such as Rbx1’s RING for cullin NEDD8ylation in vitro (Gray et al., 2002; ubiquitin, NEDD8, and SUMO is a predominant form of eukary- Kamura et al., 1999; Morimoto et al., 2003). In vivo, NEDD8yla- otic protein regulation (Kerscher et al., 2006). UBL ligation can tion is enhanced by DCN1 (Kurz et al., 2005). NEDD8 is removed alter a target’s half-life, subcellular localization, intermolecular from cullins by the COP9 Signalosome (Lyapina et al., 2001). interactions, enzymatic activity, and other functions. Generally, NEDD8 modification stimulates CRL-catalyzed ubiquitin UBLs are directed to targets by enzymatic cascades involving transfer from E2 to targets (Amir et al., 2002; Kawakami et al., an activating enzyme (E1), conjugating enzyme (E2), and 2001; Morimoto et al., 2000; Podust et al., 2000; Read et al., ligase (E3) (Capili and Lima, 2007; Dye and Schulman, 2007; 2000; Wu et al., 2000). Additionally, NEDD8ylated CRLs are not

Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. 995 recognized by CAND1 (Liu et al., 2002; Zheng et al., 2002a). De- we refer to this conformation as closed. Superposition of previ- spite much progress, mechanisms of NEDD8 modification and ous structures shows subtle differences in the relative locations CRL activation remain incompletely understood. Existing CRL of the WHB and RING subdomains (Angers et al., 2006; Golden- structures lack NEDD8 (Angers et al., 2006; Goldenberg et al., berg et al., 2004; Zheng et al., 2002b)(Figure 1A). Thus, unmod- 2004; Zheng et al., 2002b). Moreover, modeling Rbx1’s RING ified CRLs adopt a spectrum of related closed conformations. bound to E2s by docking CRL and RING-E2 structures (Hao et al., 2005; Orlicky et al., 2003; Wu et al., 2003; Zheng et al., ‘‘Open’’ Conformations of a NEDD8-Modified 2000, 2002b) raises three vexing questions. First, in a model of Cul5ctd-RING Complex Rbx1 bound to NEDD8’s E2, a predicted 35 A˚ gap between The crystals of NEDD8Cul5ctd-Rbx1 contain two complexes the E2 catalytic Cys and a cullin’s acceptor Lys begs the ques- per asymmetric unit (Figure S3). The two Rbx1 RING subdo- tion of how these residues become juxtaposed for mains adopt different relative orientations. The remainder NEDD8ylation. Second, in a model of Rbx1 bound to a ubiquitin (4HB-a/b-WHBNEDD8) superimpose well (1.3 A˚ rmsd, Figures E2, a predicted 50 A˚ gap between an E20s Cys and the sub- 1C, 1D, and S4). strate-binding site raises the question of how ubiquitin is trans- NEDD8 contacts Cul50s WHB, burying 910 A˚ 2 of exposed sur- ferred to targets. Third, the E2-to-substrate geometry is almost face area from NEDD8 and 830 A˚ 2 from Cul5 (Figures S5 and S6). certain to vary during polyubiquitination. How does CRL archi- Interactions focused around NEDD8’s C terminus culminate in tecture accommodate a succession of continually changing the isopeptide bond with Cul50s Lys724. Other interactions UBL transfer events? One way that multiple catalytic topologies involve NEDD8’s Leu8/Ile44/His68/Val70 face including all resi- could be established would be if conformational changes were dues in the Lys6-Gly10 loop, explaining the deleterious effects to accompany cullin NEDD8ylation and CRL-mediated target on CRL-mediated polyubiquitination of mutating N-terminally polyubiquitination. Here we report structural and biochemical charged residues and Leu8, Ile44, and Lys48 (Sakata et al., data revealing that NEDD8ylation influences CRL conformation, 2007; Wu et al., 2002). concomitant with activation of E3 activity. Individual NEDD8Cul5ctd-Rbx1 subdomains are structurally similar to those in unmodified CRLs. However, striking rear- RESULTS rangements in their relative positions lead to a markedly different overall conformation of the NEDD8ylated complex (Figure 1E). In Crystallization of NEDD8-Modified both NEDD8Cul5ctd-Rbx1s in the asymmetric unit, the H29 and Unmodified CRLs helix is rotated 45 about its junction with the a/b subdomain, Over 100 complexes of Cul1-5 and Rbx1-2 from numerous spe- thus repositioning the WHB relative to the 4HB and a/b subdo- cies were tested to obtain structures of both NEDD8ylated and mains, which maintain their orientation with respect to one unmodified forms of a CRL. We only obtained diffraction-quality another (Figures 1C and 1D). crystals of a NEDD8ylated CRL ctd, human NEDD8Cul5ctd- Freed from interaction with the cullin WHB, two orientations for Rbx1. Several observations indicated that this structure would the RING are observed. These result from crystal packing. One provide useful insights as a representative of the CRL superfam- RING makes fewer contacts and appears more flexible within ily: Cul5-Rbx1 is homologous to other cullin-Rbxs, NEDD8ylated the crystal, reflected by patchy protein electron density and selectively on the conserved Lys, and associated with CAND1 in high B factors (Figure S3). The two Rbx1 linkers (residues cells (Harada et al., 2002; Jones et al., 2008; Liu et al., 2002; 36–41) in the asymmetric unit extend 15 A˚ away from their Xirodimas et al., 2008)(Figures S1 and S2). Cul5-Rbx1 functions respective a/b domains, in different directions (Figure 1). As a re- as a CRL core for viral substrate adaptors (Harada et al., 2002; sult, the H29 helix and Rbx1 linker are splayed apart, such that Yu et al., 2003). Although in some cells Cul5 preferentially asso- residues in the WHB approaching Rbx1’s RING in the closed ciates with Rbx1’s close homolog Rbx2 (Kamura et al., 2004), conformation (e.g., Cul5 His763, corresponding to Cul1 Cul5 can associate with endogenous Rbx1 (Mahrour et al., Lys759, and Rbx1 Ala63) are instead widely separated in the 2008). Notably, eliminating NEDD8ylation in cells decreases modified complex, by 45 A˚ in one conformation and 72 A˚ in the Cul5-Rbx1-mediated ubiquitination (Querido et al., 2001; Yu other. The RINGs are rotated by 58 and 73, respectively, et al., 2003). Thus, we determined NEDD8Cul5ctd-Rbx1 and relative to the unmodified Cul5ctd-Rbx1 closed conformation. Cul5ctd-Rbx1 structures to directly compare modified and Superimposing NEDD8 onto the corresponding region of un- unmodified complexes for insight into the CRL family (Table S1). modified Cul5ctd-Rbx1 reveals how its ligation to cullin could induce the crystallographically observed open conformations. ‘‘Closed’’ Conformations Conserved among Unmodified In Figure S6, NEDD8Cul5ctd-Rbx1 and Cul5ctd-Rbx1 are ori- CRL ctds ented with their WHB subdomains aligned. Modeling NEDD8 In Cul5ctd-Rbx1, the 4HB, a/b, WHB, and RING subdomains are onto the corresponding site of unmodified Cul5ctd-Rbx1 reveals assembled into a single globular unit with the same arrangement that its ‘‘lower’’ portion would collide with 60% of the WHB- as in unmodified full-length CRLs, consistent with viewing this binding surface of Rbx1’s RING and that its ‘‘upper’’ portion complex as a representative CRL (Figures 1A and 1B; 1.6–2.0 would clash with 10% of the contacts between H29 and 4HB ob- A˚ rmsd) (Angers et al., 2006; Goldenberg et al., 2004; Zheng served in the closed conformation. et al., 2002b). Here, one face of the cullin WHB contacts H24 Thus, NEDD8Cul5ctd-Rbx1 adopts open conformations in and the 4HB, and the other contacts Rbx1’s RING (Figure 1B). which reorientation of the cullin WHB coincides with its separa- Due to the compact architecture from WHB-RING interactions, tion from Rbx1’s RING.

996 Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. Figure 1. Crystal Structures of Cul5ctd-Rbx1 and NEDD8Cul5ctd-Rbx1 (A) Cul5ctd (green) -Rbx1 (navy) superposition with Cul1-Rbx1 and Cul4A-Rbx1 ctds (1LDJ, olive; 1LDK, orange;, 1U6G, brown; 2HYE, pink) (Angers et al., 2006; Goldenberg et al., 2004; Zheng et al., 2002b). (B) Cartoon of Cul5ctd (green)-Rbx1 (navy), indicating positions of helices 24 and 29 (H24, H29) and the 4HB, a/b, and WHB subdomains. (C) Cartoons of the two NEDD8 (yellow, gold)Cul5ctd (lime, sky)-Rbx1 (blue, violet) complexes in the asymmetric unit, with the Cul5 Lys724NEDD8 isopeptide bond in sticks. (D) Superposition of the 4HB, a/b, and WHB subdomains for the two NEDD8Cul5ctd-Rbx1 complexes from the au, shown in three orientations, colored as in (C). (E) Superposition of the 4HB and a/b subdomains from NEDD8Cul5ctd-Rbx1s and Cul5ctd-Rbx1, oriented and colored as in (B) and (C).

NEDD8ylation Eliminates a CAND1-Binding Site In order to gain a better understanding of the Rbx1 RING prop- Comparing structures of NEDD8Cul5ctd-Rbx1 and Cul1-Rbx1- erties we used rigid body modeling, applying molecular dynam- CAND1 explains why modified CRLs do not bind CAND1. An im- ics (MD) conformational sampling. Several thousand different portant binding interface between CAND1 and Cul1-Rbx1 is conformations were produced at regular intervals along the formed by the junction of the 4HB, WHB, and RING subdomains MD trajectories, and their theoretical SAXS profiles were calcu- present in an unmodified CRL’s closed conformation (Figures 2A lated (Figure 3). Whereas poor fits were obtained for MD and 2B) (Goldenberg et al., 2004). However, this surface does not sampling of other subdomain configurations for NEDD8 exist in NEDD8ylated CRL open conformations due to NEDD8- Cul1ctd-Rbx1, good fits were obtained for conformational sam- induced reorientation of these subdomains (Figures 2C and 2D). pling of the Rbx1 linker, with the SAXS-validated three best fitting structures showing open conformations and the Rbx1 linker ex- NEDD8-Induced Conformational Opening of Cul1-Rbx1 tended, as in NEDD8Cul5ctd-Rbx1 (Figures 3C and S7). Inter- Three independent experiments indicate that NEDD8 modifi- estingly, SAXS profiles indicate that unmodified Cul1ctd-Rbx1 cation induces related conformational changes for Cul1-Rbx1. displays modest conformational flexibility relative to crystal First, we performed small-angle X-ray scattering (SAXS) analy- structures. Accordingly, prior Rbx1 crystal structures from un- ses of NEDD8Cul1ctd-Rbx1 and Cul1ctd-Rbx1 (Figures 3 modified complexes revealed multiple linker conformations and S7). The NEDD8-modified complex has a considerably (Figure S8) and very high B factors (Zheng et al., 2002b). Thus, extended maximal protein dimension (Dmax) and Rg values (cal- unmodified CRLs display moderate Rbx1 flexibility. culated based on Gunier plots) of 115 A˚ and 32.7 ± 0.1 A˚ , re- Disulfide engineering to probe WHB and RING proximity also spectively, compared with 95 A˚ and 28.0 ± 0.1 A˚ for unmodified indicates that NEDD8 induces conformational opening of full- Cul1ctd-Rbx1. The notion that NEDD8 induces large-scale con- length Cul1-Rbx1. Briefly, we engineered two independent Cys formational changes is further supported by the broadened pairs into distinct Cul1 and Rbx1 regions where Cas would be pair-distribution (P(r)) function, particularly the elongation of the within 8–10 A˚ in the closed conformation, but which are widely P(r) tail, indicating an open conformation upon NEDD8 ligation separated in the NEDD8ylated structures (Figure 4A). Consistent (Figure 3A). with closed structures, introduction of each Cys pair into

Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. 997 Figure 2. Model of a Full-Length NEDD8CRL—Elimination of a CAND1-Binding Site (A) CAND1 (red)-Cul1 (ntd, gray; 4HB, sky; a/b, lime; WHB, green)-Rbx1 (blue) structure (Goldenberg et al., 2004), shown as a cartoon and semitransparent sur- face, with the 4HB and a/b subdomains oriented as in the right view of Figure 1D. (B) Cul1-Rbx1 (Goldenberg et al., 2004; Zheng et al., 2002b), colored and oriented as in (A), highlighting the CAND1-interacting surface at the junction of the 4HB, WHB, and Rbx1 RING subdomains. (C and D) Models of full-length NEDD8CRL structures generated by superimposing the a/b- and ntd-binding 4HB subdomains from NEDD8Cul5ctd-Rbx1 and Cul1-Rbx1, oriented as in (A) and (B). NEDD8 is yellow, cullin is colored as in (A) and (B), and the two crystallographically observed Rbx1 conformations for a NEDD8-ligated complex are colored as in Figure 1C. NEDD8-mediated subdomain rearrangement would eliminate the indicated CAND1-binding surface (red circle) and position NEDD8 and its associated WHB to contact the cullin ntd (gray). unmodified Cul1-Rbx1 allows substantial intermolecular, DTT- unmodified Cul1-Rbx1 structures (Goldenberg et al., 2004; reducible disulfide crosslinking of Cul1 and Rbx1 under oxidizing Zheng et al., 2002b) and structural models of NEDD8Cul1- conditions (Figures 4B and S9). However, when oxidation is Rbx1 to identify selective protease target sites for the open performed for the paired Cys mutants of NEDD8Cul1-Rbx1, conformation. A glutamate-rich patch at the base of the H29 no significant crosslinking is observed (Figure 4B). Thus, the helix (Glu691, Glu695, and Glu697) would be differentially ex- structure of NEDD8-modified Cul1-Rbx1 is not closed. posed upon NEDD8ylation. In agreement with NEDD8-induced Proteolytic mapping also indicates NEDD8-induced confor- conformational opening, this patch is preferentially cleaved by mational opening of full-length Cul1-Rbx1. Briefly, we inspected Endoproteinase-GluC in NEDD8Cul1-Rbx1 (Figure 4C).

Figure 3. Small Angle X-Ray Scattering of NEDD8Cul1ctd-Rbx1 and Cul1ctd-Rbx1 (A) Pair-distribution (P(r)) function for NEDD8Cul1ctd-Rbx1 (blue) and Cul1ctd -Rbx1 (black). Shown are the quality of fit (c2) for individual open conformations sampled by MD and comparison of the experimental SAXS data (black) and theoretically calculated SAXS-scattering profiles for the three best fitting conforma- tions (red, blue, green) for (B) Cul1ctd-Rbx1 and (C) NEDD8Cul1ctd-Rbx1 (open–‘‘O’’). Alternative closed conformations (closed–‘‘C’’) with flexibly linked NEDD8, sampled for data on NEDD8Cul1ctd-Rbx1, are shown in olive.

998 Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. Figure 4. Probes of Conformational Change in Full-length NEDD8Cul1-Rbx1 (A) Cys pairs (A and B) at Cul1 and Rbx1 residues within 10 A˚ in the closed conformation but widely separated in open conformations based on NEDD8Cul5ctd- Rbx1 crystal structures. (B) Western blots probed with anti-Cul1 and anti-Rbx1 after disulfide crosslinking of Cul1 and Rbx1 for unNEDDylated (NEDD8) and NEDD8ylated (NEDD8) complexes harboring either wild-type (WT) or the indicated (A or B) Cys mutants of Cul1 and/or Rbx1. CysAs were in ‘‘split ‘n coexpress’’ Cul1-Rbx1, in which Cul1’s ntd and ctd are copurified as two polypeptides (Zheng et al., 2002b). Cul1 CysB masks the anti-Cul1 C terminus epitope, and thus CysBs were tested in single polypeptide Cul1 and Rbx1 from insect cells for immunodetection. (C) Endoproteinase Glu-C cleavage of a glutamate-rich patch (red) at the Cul1 hinge region in NEDD8ylated (+N8) and unNEDD8ylated (N8) full-length ‘‘split ‘n coexpress’’ Cul1-Rbx1, with reaction products detected by western blotting against Cul1 C terminus (left), NEDD8 (middle), or Cul1 ntd (right).

‘‘Freeing’’ the RING Stimulates CRL Activity characterized and their activities are assayed with well-defined, We wondered whether NEDD8-mediated enhancement of CRL purified components (Hao et al., 2005; Wu et al., 2003). In com- activity comes from freeing the Rbx1 RING from interactions parison to the activities of complexes containing non-NEDD8y- with cullin. To address this concept, we tested the effects of de- latable Cul1 harboring a Lys720Arg mutation, those lacking the leting the WHB subdomain. We utilized two well-characterized WHB showed substantially greater SCFSkp2/CksHs1-mediated CRL systems: (1) SCFbTRCP-mediated ubiquitination of a peptide polyubiquitination of phospho-p27 and SCFbTRCP-mediated from b-catenin with a single acceptor lysine (Amir et al., 2002; polyubiquitination of the b-catenin peptide (Figure 5A). Although Read et al., 2000) and (2) SCFSkp2/CksHs1-mediated ubiquitination NEDD8 attachment stimulates ubiquitination to a greater extent of phosphorylated full-length p27, which contains many ubiquitin for the b-catenin peptide, our results suggest that eliminating acceptor lysines (Morimoto et al., 2000; Podust et al., 2000). interaction between the Rbx1 RING and the WHB is activating These model CRLs offer the advantages that they are structurally for multiple SCFs. Accordingly, a WHB deletion mutant has

Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. 999 Figure 5. Functional Analysis of Mutations Influencing RING Conformational Flexibility (A) Time courses of polyubiquitination by SCFs reconstituted with fully NEDD8ylated wild-type Cul1-Rbx1 (WTN8), the nonNEDD8ylatable control (K720R), and the Cul1-Rbx1 WHB deletion mutant (DWHB). Left—SCFbTRCP-mediated polyubiquitination of a biotin-labeled b-catenin phosphopeptide, detected by western blotting with anti-biotin antisera. Right—SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27, detected by western blotting with anti-p27 antisera. (B) NEDD8ylation time course for wild-type (WT) Cul1-Rbx1, nonNEDD8ylatable (K720R) control, and variants harboring the indicated Rbx1 linker Val insertion/ deletion or Pro mutations, detected by western blotting with anti-Cul1 C terminus antisera.

1000 Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. also been found to stimulate SCFbTRCP/Cdc34-mediated poly- Asp40, and Asn41 had deleterious effects on polyubiquitination, ubiquitination of IkBa (Z.Q. Pan, personal communication). as did the Asp36 substitution (Figures 5C, 5D, and S11). Only mi- nor effects on monoubiquitin transfer were observed for these Hindering RING Orientational Flexibility Decreases mutants, suggesting that conformational restriction imposed CRL Activity by Pro substitution primarily hinders polyubiquitin chain assem- Altered Rbx1 linker orientation in unmodified CRLs could allow bly (Figures 5E and 5F). The importance of linker flexibility is juxtaposition of Ubc120s Cys and the cullin NEDD8ylation site. further substantiated by the roughly wild-type activity of linker Also, after cullin NEDD8ylation, varying RING positions could mutants harboring multiple Gly insertions either before (B) or contribute multiple catalytic geometries for CRL catalysis of after (A) Val38-Val39 and also a single Gly inserted upstream of polyubiquitination. Thus, based on modeling catalytic com- a deleterious Pro (Figures 5G–5I). plexes, we designed deletions and insertions at Rbx1 Val38- Val39 to probe roles of linker positioning and flexibility. Deletion Reoriented WHBNEDD8 Is Poised to Contact the ntd mutants test models for NEDD8ylation, as shortening the linker Full-length NEDD8-modified CRL models were generated by ctd would not allow Rbx1’s RING to position Ubc120s Cys at the superimposing NEDD8Cul5 -Rbx1 and prior full-length NEDD8ylation site. By contrast, as there appears to be some cullin-Rbx1 structures and aligned on the 4HB subdomain that slack in the linker in conformations modeled for ubiquitination, contacts the ntd. In both NEDD8ylated CRL models, the WHB insertions of different sequences would test the role of linker is positioned proximal to the ntd (Figures 2C and 2D), whereas positioning. Glycine insertions would accommodate rotation of the WHB is distal from the ntd in unmodified CRL structures the RING into a range of conformations. By contrast, extra va- (Figure 2B). Moreover, in the NEDD8ylated full-length CRL lines, which have high propensity for adopting beta structures, models, NEDD8, its linked WHB, and the 4HB together encircle would limit the RING to particular extended geometries. 3/4 of the cullin ntd (Figures 2C and 2D), with an important elec- We also designed mutants to probe conformations of individ- trostatic network on NEDD8 (Wu et al., 2002) poised to contact ual linker residues. In both NEDD8ylated complexes, residues the ntd. We speculate that subtle NEDD8-induced differences 36–38 extend similarly, with F angles ranging roughly from in the ctd-ntd interface may play a role in CRL function. 60 to 90 and J around +135 (Figure S8). By contrast, res- To test whether the WHB sequence also contributes to activa- idues 39–41 display divergent orientations. Thus, Pro substitu- tion, we assayed activities for SCFs reconstituted with two Cul1/ tions would test the importance of rotations observed about Cul5 chimeras that have homologous structures but different se- residues 39–41. Although prolines would be consistent with the quences in their predicted 4HB- and ntd-interaction surfaces structurally observed conformations of residues 36–38, if further (Figures 1 and S1). The activities of unNEDD8ylated chimeric rotations occur during catalysis a Pro substitution might be complexes generally resembled their wild-type counterparts in deleterious. three assays: (1) basal SCF ubiquitination (Figures 6A and 6B), We first tested the effects of Rbx1 linker mutations on Cul1 (2) NEDD8ylation (Figures 6CandS2), and (3) CAND1 inhibition of NEDD8ylation (Figure 5A). Linker deletions (1, 2) led to severe NEDD8ylation (Figure 6C). However, NEDD8-induced stimulation defects. Since Cul1 NEDD8ylation was not significantly affected is substantially diminished for both chimeras (Figure 6B). There- by inserting residues into the Rbx1 linker (+1, +2, and +3), we fore, sequence-dependent interactions of the relocated WHB deduce that the NEDD8ylation reaction establishes a minimum likely make important contributions to NEDD8-mediated activa- Rbx1 linker extension. Consistent with Rbx1 linker rotation, Pro tion. Additional studies are required to provide detailed insights substitutions for Ile37 and Val39 also impeded NEDD8ylation. into structural remodeling of a full-length NEDD8ylated CRL. To test effects of mutations on CRL-mediated ubiquitination, we purified fully NEDD8ylated forms for all the Cul1-Rbx1 mu- Order of UBL Targeting Is an Inherent CRL Property, tants, except for the Val38-Val39 double deletion that could Independent of E2 and UBL Identity not be substantially NEDD8ylated even with attempts to force Differences between the Culctd-Rbx1 and NEDD8Culctd-Rbx1 the reaction. Linker extension length plays an important role in structures raise the possibility that conformations may influence SCF activity, as ubiquitination is substantially impaired with the selection between cullin and substrate modification. This notion Val insertion mutations (Figures 5C–5F, S10, and S11). However, is consistent with results obtained while establishing our ubiqui- there may be differences in geometries assumed by the different tination assays. In comparing wild-type unNEDD8ylated Cul1 insertion mutants since there is not a linear correlation between and nonNEDD8ylatable Cul1Lys720Arg in SCF assays, we ob- the number of inserted residues and effects on monoubiquitina- serve that (1) Cul1 itself is monoubiquitinated in a Lys720-depen- tion of the b-catenin peptide (Figures 5E and 5F). Also consistent dent manner, (2) Cul1 monoubiquitination precedes ubiquitin with the crystal structures, Pro substitutions in place of Val39, transfer to phospho-p27 and b-catenin peptide, and (3) ubiquitin

(C) SCFbTRCP-mediated polyubiquitination of a biotin-labeled b-catenin phosphopeptide or (D) SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27 for fully NEDD8ylated SCFs harboring the indicated Rbx1 linker mutants. Pulse-chase Ub(K > R) transfer from UbcH5b for (E) SCFbTRCP and a biotin-labeled b-catenin phosphopeptide and (F) SCFSkp2/CksHs1 and phospho-p27. (G) NEDD8ylation time course for split ‘n coexpress Cul1-Rbx1 and for Rbx1 linker triple Val or Gly insertions (left) and indicated Pro mutants or those with up- stream Gly insertions ( G) (right). (H) SCFbTRCP-mediatedˆ polyubiquitination of a biotin-labeled b-catenin phosphopeptide and (I) SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27 for G fully NEDD8ylated SCFs harboring Rbx1 linker triple Val or Gly insertions (left) and indicated Pro mutants or those with upstream Gly insertions (ˆ ) (right).

Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. 1001 Deshaies, 2005; Tang et al., 2005). Studies of prototypic mem- bers of the SCF family indicate that each CRL may ubiquitinate tens of substrates, often on multiple acceptor lysines. Further- more, SCFs containing structurally similar F-box protein sub- strate adaptors display substantial differences in their optimal degron-to-acceptor distances. These can vary by tens of resi- dues even among substrates of a given SCF. Thus, the mechanis- tic requirements for CRL-mediated ubiquitination are complex. Our data indicate that CRL conformational variability con- tributes to multiple catalytic functions (Figure 7B), including NEDD8ylation (Figure 7C), and target polyubiquitination (Figures 7D and 7E). The previously observed closed structures of unmod- ified CRLs appear to represent the ‘‘off’’ state, lacking active geometries for ligation of NEDD8 (or in vitro, ubiquitin) to cullin, or for target polyubiquitination. Structural variability observed in crystal structures and SAXS points to some flexibility within the closed form, which is maintained in its inactive state in cells by as- sociation with CAND1 (Goldenberg et al., 2004). Upon adaptor binding and CAND1 dissociation (Bornstein et al., 2006), possibly with influences from E2UBL binding, unmodified CRLs adopt conformations that favor cullin modification (Figure 7C). NEDD8 ligation shifts the equilibrium to favor flexible open conformations that prefer adaptor-bound substrates. Open CRLs would remain active and unable to bind CAND1 (Figure 2) until deNEDD8ylation. Interestingly, CRLs may display differences in intrinsic flexibil- ity. A kink in Cul4A’s H29 helix subtly displaces the WHB and RING relative to other CRLs (Angers et al., 2006). Further, S. cer- evisiae Cdc53p has a unique Gly-rich insertion immediately preceding the WHB (Figure S12), at the hinge we observe struc- turally, which may impart flexibility. Figure 6. NEDD8-Dependent Role of the WHB Sequence Despite conformational flexibility, the relative location of the Time courses of polyubiquitination by (A) SCFbTRCP and (B) SCFSkp2/CksHs1 RING is important. Insertion of a flexible hinge between Cul1’s reconstituted with fully NEDD8ylated wild-type Cul1-Rbx1 (WTN8) or Cul1/5 ntd and ctd destroys ubiquitination (Zheng et al., 2002b), and chimeras (A or BN8), and their nonNEDD8ylatable counterparts (K > R). the extension length of the linker tethering the RING is tightly con- (C) NEDD8ylation time course and CAND1 inhibition for wild-type Cul1-Rbx1 strained by a minimum required for Cul1 NEDD8ylation and and the Cul1/5-Rbx1 chimeras. Reactions were separated by SDS-PAGE a maximum limit for SCF-mediated polyubiquitination (Figure 5). and visualized by Coomassie staining. In addition to the RING, the WHB is also an effector of NEDD8 ac- tivation, as evidenced by mutagenesis (Figure 6). Although further ligation to Cul1 activates SCFs (Figure 7A). Consistent with our structural studies of full-length modified CRLs will be required to findings, in a study lacking NEDD8ylation components all active understand functions of the relocated WHB, we note that in the SCFs contained ubiquitin-modified Cul1 (Wu et al., 2003). Fur- NEDD8Cul5ctd-Rbx1 complexes, both the WHB and NEDD8 thermore, in the absence of NEDD8, the yeast cullin Rtt101p is are poised to interact with the cullin ntd. We speculate that the apparently modified by ubiquitin or another UBL (Laplaza et al., WHB and/or NEDD8 may alter the conformation of the ctd-ntd 2004). Although cullin NEDD8ylation is generally far more effi- junction in a way that contributes to ubiquitin E3 activity. cient than ubiquitination (data not shown), our results demon- Many other aspects of CRL function, such as association with strate that irrespective of the UBL (NEDD8 or ubiquitin) or E2 DCN1 during NEDD8ylation (Kurz et al., 2005) or the deNEDD8y- (Ubc12 or UbcH5), cullin is the preferred first target of a single lating COP9 Signalosome (Lyapina et al., 2001), could also be UBL. UBL ligation to Cul1 switches the target preference to sub- under conformational control. CRL activity may also be influ- strate and activates substrate polyubiquitination. We propose enced by changes to substrate structures upon their phosphor- that the conformation of an unmodified CRL favors cullin as ylation, noncovalent association with partners, and/or ubiquitin the first UBL target, and this modification repositions the cullin ligation. Conformations associated with E2 ‘‘backside’’ binding WHB and frees the Rbx1 RING to favor target polyubiquitination. to UBLs and structural differences between polyubiquitin moie- ties dictated by linkage and chain length may also influence CRL DISCUSSION activities (Brzovic et al., 2006; Pickart and Fushman, 2004; Sakata et al., 2007). Hundreds of CRLs, generated by the combination of several cullin Conformational variation is emerging as a widespread theme subunits with numerous substrate adaptors, catalyze a stunning in UBL conjugation cascades. For example, E1s and HECT array of reactions. (Cardozo and Pagano, 2004; Petroski and E3s undergo domain rotations of greater than 100 that are

1002 Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. Figure 7. Conformational Control of CRL Activities (A) Polyubiquitination reactions with SCFbTRCP/b-catenin phosphopeptide (left) and SCFSkp2/CksHs1/phospho-p27 (right) reconstituted with nonNEDD8ylatable (K720R), unNEDD8ylated wild-type (WT), and fully NEDD8ylated Cul1-Rbx1 (WTN8). Reaction products were detected by immunoblotting, top panels with anti-biotin (left) or anti-p27 (right), middle with anti-His (ubiquitin; green *–Cul1Ubiquitin), and lower with anti-Cul1 C terminus antisera. (B–E) Models of conformations involved in CRL activities. (B) Structural model of SCFbTRCP-b-catenin phosphopeptide complex (Wu et al., 2003; Zheng et al., 2002b) with Rbx1 RING and E2 docked based on the c-Cbl-UbcH7 structure (Zheng et al., 2000), with the generic E2 in light blue, the E2’s catalytic Cys as a yellow sphere, Rbx1 in blue, the ctd portion of Cul1 in green, Cul1 ntd in gray, the substrate adaptor Skp1-F-box protein (Fbp) bTRCP complex in purple and magenta, and b–catenin peptide target in green sticks. Approximate distances to be spanned for NEDD8 transfer to the cullin target Lys (green stick) and for ubiquitin trans- fer to target during polyubiquitination are indicated with arrows. (C) Model of cullin ligation to NEDD8 (yellow), where the NEDD8 E2 (cyan) Cys and cullin target lysine are justaposed via rotation of the Rbx1 linker (residues 36–41). (D) Model of ubiquitin (orange) transfer from ubiquitin E2 (teal) to target by a NEDD8ylated CRL, generated by superimposing the a/b- and ntd-binding 4HB subdomains from NEDD8Cul5ctd-Rbx1 and Cul1-Rbx1. The E2 Cys and peptide target were brought into proximity via rotation of the Rbx1 linker. (E) Model of target polyubiquitination by a NEDD8ylated CRL, where rotation about the Rbx1 linker allows multiple catalytic geometries associated with polyubiquitin chain extension. important for catalysis (Huang et al., 2005, 2007; Verdecia et al., UBA3, Ubc12, NEDD8, Skp1-Skp2, Skp1-bTRCP, CksHs1, CAND1, and Cy- 2003). Also, analogous to freeing the Rbx1 RING, the Itch and clinA-cdk2 were prepared as described (Brown et al., 1999; Goldenberg Smurf2 E3s are activated by freeing their HECT domains from et al., 2004; Huang et al., 2007, 2008; Schulman et al., 2000; Wu et al., 2003). Most assays used ‘‘split ‘n coexpress’’ Cul1-Rbx5-108 (and variants), autoinhibitory intramolecular interactions (Gallagher et al., shown previously to be structurally and biochemically identical to single poly- 2006; Wiesner et al., 2007). Similarly, the RING E3 Ubr1 is peptide Cul1-Rbx1 (Zheng et al., 2002b). Ctds are Cul5401-780 or Cul1411-776, released from an autoinhibited state that prevents access to its with solubilizing substitutions of ntd-binding 4HB hydrophobics (Cul5 substrate-binding site (Du et al., 2002). L407E, L439K, V440K; Cul1 L421E, V451E, V452K, Y455K) (Zheng et al., As revealed from structures of other signaling proteins com- 2002b). Culctd-Rbx1s coexpressed as GST-Culctd-MBP-Rbx1 were purified ctd posed of multiple subdomains, the modularity of enzymes pres- by glutathione affinity. GSTCul -MBPRbxs were NEDD8ylated in 30 mM Tris-Cl, 50 mM NaCl, 10 mM MgCl, 10 mM ATP, pH 8.0 for 5 hr at 4C with ent in UBL cascades probably amplifies their opportunity for 0.043 APPBP1-UBA3 and Ubc12, 2.53 NEDD8. After TEV protease treat- conformational control. It seems likely that UBL attachment to ment, Culctd-Rbxs were purified by cation exchange and gel filtration, concen- other modular enzymes could drive reaction cascades by shift- trated to 10–15 mg/ml in 25 mM HEPES or Tris-Cl, 150 mM NaCl, 5 mM DTT, ing conformational equilibria to disfavor inactive architectures pH 7.0 or 7.6, aliquotted, flash-frozen, and stored at 80C. For crystallogra- and favor subdomain rearrangements that promote subsequent phy, NEDD8 Leu62SeMet was used, as the additional Se facilitates structure activities. determination without affecting function (D. Huang and B.A.S., unpublished data). For biochemistry, 8 mM Cul1-Rbx1 (and variants) were NEDD8ylated in 50

EXPERIMENTAL PROCEDURES mM Tris, 150 mM NaCl, 2.5 mM MgCl2, 1.25 mM ATP, pH 7.6 with 700 nM APPBP1-UBA3, 1.8 mM Ubc12, 60 mM NEDD8 in 50 mM Tris, 150 mM NaCl,

Constructs, Protein Preparation, and Antibodies 2.5 mM MgCl2, 1.25 mM ATP, pH 7.6 for 30 min at room temperature (RT) Human proteins were generated by standard molecular techniques, with con- (higher enzyme concentrations/longer reaction times for some variants). After struct-coding sequences entirely verified. Ube1, UbcH5b, ubiquitin, APPBP1- adding DTT to 10 mM, NEDD8ylated Cul-Rbxs were purified by gel filtration.

Cell 134, 995–1006, September 19, 2008 ª2008 Elsevier Inc. 1003 GST-p27 (the k-version that can be phosphorylated on T187 by cdk2, (Vlach conditions. For pulse-chase assays, 8.3 mM UbcH5b was loaded for 25 min at et al., 1997; also with the S10A mutation that restricts phosphorylation to RT with 230 nM Ube1, 65 mM Ub(K > R) in 100 mM HEPES, 100 mM NaCl,

T187; Hao et al., 2005) was purified by glutathione affinity, treated with throm- 1 mg/ml BSA, 5 mM MgCl2, 1 mM ATP, pH 7.6, and quenched with 50 mM bin, and purified to homogeneity by gel filtration. Cdc34BKR (a Lys > Arg ver- EDTA for 5 min on ice. 1.2 mM UbcH5bUb(K > R) thioester conjugate was sion that cannot be inhibited by autoubiquitination; Scaglione et al., 2007) was diluted into chase mixes of 0.5 mM SCF, 0.5 mM phospho-p27-CyclinA- purified similarly. Four micromolar p27 was phosphorylated on T187 at 30C Cdk2, or 5 mM b-catenin phosphopeptide, 75 mM Tris, 100 mM NaCl, 50 for 30 min at 1:1 with CyclinA-cdk2 in 40 mM Tris-HCl, 10 mM MgCl2,1mM mM EDTA, 1 mg/ml BSA, pH 7.6, at 4 C(b-cat) or RT (p27). Ub reactions ATP, 1 mM DTT, pH 7.6. b-catenin phosphopeptide (Wu et al., 2003) has were stopped with DTT buffer and separated by SDS-PAGE, and products a C-terminal biotin attached via a PEG linker, which we found does not influ- were detected by immunoblotting. ence function (DCS and BAS, not shown). Antibodies used for westerns For disulfide crosslinking, Cys pair A is Cul1K759C-Rbx1A63C (Figure S1), in were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 ‘‘split ‘n coexpress’’ Cul1-Rbx1 (Zheng et al., 2002b). An additional Cul1 (Rockland 100-401-A13), His-tag (QIAGEN, 34660), p27 (Santa Cruz, sc- C752A mutation (also in controls) minimized background crosslinking to this 527,sc-16324), and biotin (Rockland, 100-4198). DWHB mutant terminates Cul1 Cys. Cys pair B is Cul1 with a C-terminal Gly-Cys extension (+2Cys) after Cul1 residue 692. The chimera junctions are at Cul1/Cul5 residues and Rbx1 L88C. Cys pair B and controls were expressed in insect cells as de- 697/701 (A) and 707/711 (B) (Figure S1). scribed (Zheng et al., 2002b) with Cul1 as a single polypeptide for immunode- tection. Oxidation was for 10 s at RT with 10 mM NatT and 15 mM Cul1-Rbx1 Crystal Structure Determination and Cys variants, quenched for 5 min with 20 mM NEM. Disulfide-crosslinked Cul5ctd-Rbx1 crystals were grown by microseeding 4C hanging drops 1:1 Cul1 and Rbx1 separated from noncrosslinked proteins by nonreducing with 2% PEG3350, 0.1 M HEPES, 0.2 M L-Pro, pH 8.0, in P212121 with a = SDS-PAGE (Figure 4) or after reduction by DTT (Figure S9). 63.0, b = 65.5, c = 141.1 and one complex per au and frozen in 5% Limited proteolysis was performed in PBS at RT with 7 mM Cul1-Rbx1 or PEG3350, 0.1 M HEPES, 0.2 M L-Pro, 8% glycerol, 8% ethylene glycol, 8% NEDD8Cul1-Rbx1 and 21 mM Endoproteinase Glu-C. Mass spectrometry sucrose, pH 8.0. NEDD8Cul5ctd-Rbx1 crystals were grown by microseeding and Edman degradation are consistent with initial cleavage at Cul1 Glu691, 4 C hanging drops 1:1 with 19% PEG3350, 275 mM (NH4)2PO4, 5 mM DTT in Glu695, and Glu697.

P212121 with a = 88.2, b = 122.4, c = 128.6 and two complexes per au and fro- zen in 19% PEG3350, 250 mM (NH4)2PO4, 10 mM DTT, 8% glycerol, 8% eth- ACCESSION NUMBERS ylene glycol, 8% sucrose. Data were processed with HKL2000 (Otwinowski ctd and Minor, 1997). SAD phases for NEDD8Cul5 -Rbx1 were obtained with Coordinates/structure factor amplitudes have been deposited in the Protein SOLVE with 24 of 36 Ses, with hand determination/density modification by RE- Data Bank with accession codes 3DPL and 3DQV. SOLVE (Terwilliger and Berendzen, 1999). Initial maps showed continuous density for NEDD8, Cul5ctd, and Rbx1 19-35. Rbx1’s RINGs were built by boot- SUPPLEMENTAL DATA strapping in O (Jones et al., 1991). Anomalous data collected at 1.283 A˚ showed zincs and confirmed RING positions. Cul5ctd-Rbx1 structure was de- Supplemental Data include one table and twelve figures and can be found with termined by molecular replacement using PHASER (Storoni et al., 2004) with this article online at http://www.cell.com/cgi/content/full/134/6/995/DC1/. 4HB, a/b, and WHB subdomains from NEDD8Cul5ctd-Rbx1. SeMet anoma- lous difference fourier maps from data collected at 0.98 A˚ confirm SeMet and ACKNOWLEDGMENTS zinc positions. Models were refined with CNS (Bru¨ nger et al., 1998) and contain Cul5ctd-Rbx1 Cul5 401–517 and 520–780 and Rbx1 19–63 and 67–108; We are grateful to D. King and S. Zhou for mass spectrometry/Edman se- NEDD8Cul5ctd-Rbx11 all NEDD8 residues, Cul5 401-515 and 520-780, quencing, J. Endicott for cdk2 plasmid, C. Ross and Staff at ALS 8.2.1, ALS and Rbx1 20-105; NEDD8Cul5ctd-Rbx12 all NEDD8 residues, Cul5 401– 12.3.1, and NSLS X25 beamlines, V. Pagala, P. Murray, R. Deshaies, Z.Q. 517 and 520–780, and Rbx1 19–50 and 53–105, with 58–60, 63–65, 71, 78, Pan, J.W. Harper, M. Lee, B. Dye, D. Miller, S. Bozeman, and members of and 93–94 built into patchy density and thus assigned zero occupancy. Details ctd the Schulman lab for assistance, advice, and/or discussions. ALS is supported are in Table S1. We presume the Rfree for NEDD8Cul5 -Rbx1 reflects high by U.S. DOE Contract No. DE-AC02-05CH11231, and NSLS by DE-AC02- RING mobility. 98CH10886. This work was supported in part by American Lebanese Syrian Associated Charities of St. Jude, by NIH P30CA021765 to St. Jude, the SAXS Howard Hughes Medical Institute, a Beckman Young Investigator Award, The P(r), calculated with GNOM (Svergun, 1992), for Cul1ctd-Rbx1 was indica- a Pew Scholar Award, the Phillip and Elizabeth Gross Foundation, the NIH tive of a globular particle whereas NEDD8Cul1ctd-Rbx1 showed a shifted (R01GM069530), and DOD (DAMD17-03-0420) to B.A.S. and by an American maximum and extended tail. The NEDD8Cul1ctd-Rbx1 sample was properly Cancer Society fellowship to D.M.D. B.A.S. is an Investigator of the Howard folded as revealed by the Kratky plot (Kratky and Porod, 1949) indicating the Hughes Medical Institute. extended nature of this sample with respect to Cul1ctd-Rbx1. Crystal struc- tures of Cul1ctd-Rbx1 and NEDD8Cul5ctd-Rbx1 were used in rigid body mod- Received: March 10, 2008 eling applying MD as described (Hammel et al., 2005, 2007). Additional details Revised: June 23, 2008 are in Figure S7. Accepted: July 15, 2008 Published: September 18, 2008 Biochemical Assays NEDD8ylation was assayed with 500 nM APPBP1-UBA3, 1.2 mM Ubc12, and REFERENCES 4 mM Cul1-Rbx1 and variants, initiated with 60 mM NEDD8 in 50 mM Tris-HCl, 150 mM NaCl, 2.5 mM MgCl , 1.25 mM ATP, pH 7.6. 1:1 complexes with 2 Amir, R.E., Iwai, K., and Ciechanover, A. (2002). The NEDD8 pathway is essen- CAND1 were used in Figure 6. Reactions were stopped with SDS sample tial for SCF(b-TrCP)-mediated ubiquitination and processing of the buffer, and products were separated by SDS-PAGE, and visualized by Coo- NF-kappa B precursor p105. J. Biol. Chem. 277, 23253–23259. massie or western. Polyubiquitination was assayed at RT with 150 nM Ube1, UbcH5b (1 mMinFigures 5 and 7 and 4 mM in Figures 5A and 6), 75 or 200 Angers, S., Li, T., Yi, X., MacCoss, M.J., Moon, R.T., and Zheng, N. (2006). nM SCF (b-catenin or p27), and 10 mM b-catenin phosphopeptide or 0.2 mM Molecular architecture and assembly of the DDB1–CUL4A ubiquitin ligase phospho-p27-CyclinA-Cdk2 in 75 mM Tris-HCl, 100 mM NaCl, 1 mg/ml machinery. Nature 443, 590–593.

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