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Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Cell. Author manuscript; available in PMC Mar 19, 2009.
Published in final edited form as:
PMCID: PMC2628631
HHMIMSID: HHMIMS71581

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

SUMMARY

Cullin-RING Ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8~Cul5ctd-Rbx1 and SAXS analysis of NEDD8~Cul1ctd-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

INTRODUCTION

Covalent attachment of ubiquitin-like proteins (UBLs) such as ubiquitin, NEDD8, and SUMO is a predominant form of eukaryotic protein regulation (Kerscher et al., 2006). UBL ligation can alter a target’s half-life, subcellular localization, intermolecular interactions, enzymatic activity, and other functions. Generally, UBLs are directed to targets by enzymatic cascades involving an activating enzyme (E1), conjugating enzyme (E2), and ligase (E3) (Capili and Lima, 2007; Dye and Schulman, 2007; Knipscheer and Sixma, 2007). In humans, UBL ligation is catalyzed by ~600 potential E3s (Li et al., 2008). E3 activity is generally regulated by post-translational modifications such as phosphorylation, hydroxylation, processing, or UBL-attachment.

The largest E3 subclass consists of modular, multisubunit Cullin-RING ligases (CRLs) (Cardozo and Pagano, 2004; Petroski and Deshaies, 2005). Human CRL cores contain a cullin (Cul1, 2, 3, 4A, 4B, 5) and Rbx/Roc1 or 2, and adopt an elongated architecture with substrate- and E2-binding sites at opposite ends (Zheng et al., 2002b). At one end, a cullin’s N-terminal domain (ntd) can pair with up to hundreds of substrate-binding adaptors. Distally, the C-terminal domain (ctd) is assembled from 4HB, α/β-, and WHB subdomains. The 4HB interacts with the ntd. The α/β-subdomain binds Rbx1’s N-strand. Rbx1’s RING, which is thought to bind E2s in the same manner as other E3 RINGs (Zheng et al., 2000), is connected to Rbx1’s N-strand via a 6-residue linker. In unmodified CRLs, Rbx1’s RING contacts a cullin’s WHB (Zheng et al., 2002b).

CRL activity is regulated by both inhibition and activation. Cul1, -2, -3, -4A, and -5 bind CAND1, the CRL assembly inhibitor (Liu et al., 2002; Zheng et al., 2002a). A Cul1-Rbx1-CAND1 structure showed the two CRL ends as key CAND1 binding sites (Goldenberg et al., 2004). Subtle differences in the relative locations of Cul1’s N- and C-termini observed upon CAND1 binding indicate potential for altered orientations of a CRL’s ntd and 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) (Jones et al., 2008; Pan et al., 2004; Xirodimas et al., 2008). As with ubiquitination cascades, NEDD8’s E2 (Ubc12) binds Rbx1’s RING for cullin NEDD8ylation in vitro (Gray et al., 2002; Kamura et al., 1999; Morimoto et al., 2003). In vivo, NEDD8ylation is enhanced by DCN1 (Kurz et al., 2005). NEDD8 is removed from cullins by the COP9 Signalosome (Lyapina et al., 2001).

NEDD8 modification stimulates CRL-catalyzed ubiquitin transfer from E2 to targets (Amir et al., 2002; Kawakami et al., 2001; Morimoto et al., 2000; Podust et al., 2000; Read et al., 2000; Wu et al., 2000). Additionally, NEDD8ylated CRLs are not recognized by CAND1 (Liu et al., 2002; Zheng et al., 2002a). Despite much progress, mechanisms of NEDD8 modification and CRL activation remain incompletely understood. Existing CRL structures lack NEDD8 (Angers et al., 2006; Goldenberg et al., 2004; Zheng et al., 2002b). Moreover, modeling Rbx1’s RING 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., 2002b; Zheng et al., 2000) raises three vexing questions. First, in a model of Rbx1 bound to NEDD8’s E2, a predicted ~35 Å gap between the E2 catalytic Cys and a cullin’s acceptor Lys begs the question of how these residues become juxtaposed for NEDD8ylation. Second, in a model of Rbx1 bound to a ubiquitin E2, a predicted ~60Å gap between an E2’s Cys and the substrate binding site raises the question of how ubiquitin is transferred to targets. Third, the E2-to-substrate geometry is almost certain to vary during polyubiquitination. How does CRL architecture accommodate a succession of continually changing UBL transfer events? One way that multiple catalytic topologies could be established would be if conformational changes were to accompany cullin NEDD8ylation and CRL-mediated target polyubiquitination. Here we report structural and biochemical data revealing that NEDD8ylation influences CRL conformation, concomitant with activation of E3 activity.

RESULTS

Crystallization of NEDD8-modified and unmodified CRLs

Over 100 complexes of Cul1-5 and Rbx1-2 from numerous species were tested to obtain structures of both NEDD8ylated and unmodified forms of a CRL. We only obtained diffraction-quality crystals of a NEDD8ylated CRL C-terminal domain, human NEDD8~Cul5ctd-Rbx1. Several observations indicated that this structure would provide useful insights as a representative of the CRL superfamily: Cul5-Rbx1 is homologous to other cullin-Rbxs, NEDD8ylated selectively on the conserved Lys, and associated with CAND1 in cells (Harada et al., 2002; Jones et al., 2008; Liu et al., 2002; Xirodimas et al., 2008) (Figure S1, S2). Cul5-Rbx1 functions as a CRL core for viral substrate adaptors (Harada et al., 2002; Yu et al., 2003). Although in some cells Cul5 preferentially associates with Rbx1’s close homolog Rbx2, Cul5 can associate with endogenous Rbx1 (Kamura et al., 2004; Mahrour et al., 2008). Notably, eliminating NEDD8ylation in cells decreases Cul5-Rbx1-mediated ubiquitination (Querido et al., 2001; Yu et al., 2003).Thus, we determined NEDD8~Cul5ctd-Rbx1 and Cul5ctd-Rbx1 structures to directly compare modified and unmodified complexes for insight into the CRL family (Table S1).

“Closed” conformations conserved among unmodified CRL ctds

In Cul5ctd-Rbx1, the 4HB, α/β, WHB, and RING subdomains are assembled into a single globular unit with the same arrangement as in unmodified full-length CRLs, consistent with viewing this complex as a representative CRL (Figure 1A, B; 1.6–2.0 Å rmsd) (Angers et al., 2006; Goldenberg et al., 2004; Zheng et al., 2002b). Here, one face of the cullin WHB contacts H24 and the 4HB, and the other contacts Rbx1’s RING (Figure 1B). Due to the compact architecture from WHB-RING interactions, we refer to this conformation as “closed”. Superposition of previous structures shows subtle differences in the relative locations of the WHB and RING subdomains (Angers et al., 2006; Goldenberg et al., 2004; Zheng et al., 2002b) (Figure 1A). Thus, unmodified CRLs adopt a spectrum of related closed conformations.

Figure 1
Crystal structures of Cul5ctd-Rbx1 and NEDD8~Cul5ctd-Rbx1

“Open” conformations of a NEDD8-modified Cul5ctd - RING complex

The crystals of NEDD8~Cul5ctd-Rbx1 contain two complexes per asymmetric unit (Figure S3). The two Rbx1 RING subdomains adopt different relative orientations. The remainder (4HB-α/β-WHB~NEDD8) superimpose well (1.3 Å rmsd, Figure 1C, 1D, Figure S4).

NEDD8 contacts Cul5’s WHB, burying 910 Å2 of exposed surface area from NEDD8 and 830 Å2 from Cul5 (Figures S5, S6). Interactions focused around NEDD8’s C-terminus culminate in the isopeptide bond with Cul5’s Lys724. Other interactions involve NEDD8’s Leu8/Ile44/His68/Val70 face including all residues in the Lys6-Gly10 loop, explaining the deleterious effects of mutating N-terminal charged residues, Leu8, Ile44, and Lys48 on CRL-mediated polyubiquitination (Sakata et al., 2007; Wu et al., 2002).

Individual NEDD8~Cul5ctd-Rbx1 subdomains are structurally similar to those in unmodified CRLs. However, striking rearrangements in their relative positions lead to a markedly different overall conformation of the NEDD8ylated complex (Figure 1E). In both NEDD8~Cul5ctd-Rbx1s in the asymmetric unit, the H29 helix is rotated ~45° about its junction with the α/β subdomain, thus repositioning the WHB relative to the 4HB and α/β subdomains, which maintain their orientation with respect to one another (Figure 1C, 1D).

Freed from interaction with the cullin WHB, two orientations for the RING are observed. These result from crystal packing. One RING makes fewer contacts and appears more flexible within the crystal, reflected by patchy protein electron density and high B-factors (Figure S3). The two Rbx1 linkers (residues 36–41) in the asymmetric unit extend ~15 Å away from their respective α/β domains, in different directions (Figure 1). As a result, the H29 helix and Rbx1 linker are splayed apart, such that residues in the WHB approaching Rbx1’s RING in the closed conformation (e.g. Cul5 His763, corresponding to Cul1 Lys759, and Rbx1 Ala63) are instead widely separated in the modified complex, by 45 Å in one conformation and 72 Å in the other. The RINGs are rotated by~58° and ~73°, respectively, relative to the unmodified Cul5ctd-Rbx1 closed conformation.

Superimposing NEDD8 onto the corresponding region of unmodified Cul5ctd-Rbx1 reveals how its ligation to cullin could induce the crystallographically-observed open conformations. In Figure S6, NEDD8~ Cul5ctd-Rbx1 and Cul5ctd-Rbx1 are oriented with their WHB subdomains aligned. Modeling NEDD8 onto the corresponding site of unmodified Cul5ctd-Rbx1 reveals that its “lower” portion would collide with ~60% of the WHB-binding surface of Rbx1’s RING and that its “upper” portion would clash with 10% of the contacts between H29 and 4HB observed in the closed conformation.

Thus, NEDD8~Cul5ctd-Rbx1 adopts “open” conformations in which reorientation of the cullin WHB coincides with its separation from Rbx1’s RING.

NEDD8ylation eliminates a CAND1-binding site

Comparing structures of NEDD8~Cul5ctd-Rbx1 and Cul1-Rbx1-CAND1 explains why modified CRLs do not bind CAND1. An important binding interface between CAND1 and Cul1-Rbx1 is formed by the junction of the 4HB, WHB, and RING subdomains present in an unmodified CRL’s closed conformation (Figure 2A, B)(Goldenberg et al., 2004). However, this surface does not exist in NEDD8ylated CRL open conformations, due to NEDD8-induced reorientation of these subdomains (Figure 2C, D).

Figure 2
Model of a full-length NEDD8~CRL – elimination of a CAND1 binding site

NEDD8-induced conformational opening of Cul1-Rbx1

Three independent experiments indicate NEDD8 modification induces related conformational changes for Cul1-Rbx1. First, we performed Small Angle X-ray Scattering (SAXS) analyses of NEDD8~Cul1ctd-Rbx1 and Cul1ctd-Rbx1 (Figure 3, S7). The NEDD8-modified complex has a considerably extended maximal protein dimension (Dmax) and Rg values (calculated based on Gunier plots) of ~115 Å and 32.7 ±0.1 Å, respectively, compared with ~95Å and 28.0 ±0.1 Å for unmodified Cul1ctd-Rbx1. The notion that NEDD8 induces large-scale conformational changes is further supported by the broadened P(r) function, particularly the elongation of the P(r) tail, indicating an open conformation upon NEDD8 ligation (Figure 3A).

Figure 3
Small Angle X-Ray Scattering of NEDD8~Cul1ctd-Rbx1 and Cul1ctd-Rbx1

In order to gain a better understanding of the Rbx1 RING properties we used rigid body modeling, applying Molecular Dynamics (MD) conformational sampling. Several thousand different conformations were produced at regular intervals along the MD trajectories, and their theoretical SAXS profiles were calculated (Figure 3). Whereas poor fits were obtained for MD sampling of other subdomain configurations for NEDD8~Cul1ctd-Rbx1, good fits were obtained for conformational sampling of the Rbx1 linker, with the SAXS-validated three best fitting structures showing open conformations and the Rbx1 linker extended, as in NEDD8~Cul5ctd-Rbx1 (Figure 3C, S7). Interestingly, SAXS profiles indicate that unmodified Cul1ctd-Rbx1 displays modest conformational flexibility relative to crystal structures. Accordingly, prior Rbx1 crystal structures from unmodified complexes revealed multiple linker conformations (Figure S8) and very high B-factors (Zheng et al., 2002b). Thus, unmodified CRLs display moderate Rbx1 flexibility.

Disulfide engineering to probe WHB and RING proximity also indicates that NEDD8 induces conformational opening of full-length Cul1-Rbx1. Briefly, we engineered two independent Cys pairs into distinct Cul1 and Rbx1 regions where Cαs would be within 8–10 Å in the closed conformation, but which are widely separated in the NEDD8ylated structures (Figure 4A). Consistent with closed structures, introduction of each Cys pair into unmodified Cul1-Rbx1 allows substantial intermolecular, DTT-reducible disulfide crosslinking of Cul1 and Rbx1 under oxidizing conditions (Figure 4B, S9). However, when oxidation is performed for the paired Cys mutants of NEDD8~Cul1-Rbx1, no significant crosslinking is observed (Figure 4B). Thus, the structure of NEDD8-modified Cul1-Rbx1 is not closed.

Figure 4
Probes of Conformational Change in Full-length NEDD8~Cul1ctd-Rbx1

Proteolytic mapping also indicates NEDD8-induced conformational opening of full-length Cul1-Rbx1. Briefly, we inspected unmodified Cul1-Rbx1 structures (Goldenberg et al., 2004; Zheng et al., 2002b) and structural models of NEDD8~Cul1-Rbx1 to identify selective protease target sites for the open conformation. A glutamate-rich patch at the base of the H29 helix (Glu691, Glu695, and Glu697) would be differentially exposed upon NEDD8ylation. In agreement with NEDD8-induced conformational opening, this patch is preferentially cleaved by Endoproteinase-GluC in NEDD8~Cul1-Rbx1 (Figure 4C).

“Freeing” the RING stimulates CRL activity

We wondered whether NEDD8-mediated enhancement of CRL activity comes from “freeing” the Rbx1 RING from interactions with cullin. To address this concept, we tested the effects of deleting the WHB subdomain. We utilized two well-characterized CRL systems: (1) SCFβTRCP-mediated ubiquitination of a peptide from β-catenin with a single acceptor lysine (Amir et al., 2002; Read et al., 2000) and (2) SCFSkp2/CksHs1-mediated ubiquitination of phosphorylated full-length p27, which contains many ubiquitin acceptor lysines (Morimoto et al., 2000; Podust et al., 2000). These model CRLs offer the advantages that they are structurally characterized and their activities are assayed with well-defined, purified components (Hao et al., 2005; Wu et al., 2003). In comparison to the activities of complexes containing non-NEDD8ylatable Cul1 harboring a Lys720Arg mutation, those lacking the WHB showed substantially greater SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27 and SCFβTRCP-mediated polyubiquitination of the β-catenin peptide (Figure 5A). Although NEDD8-attachment stimulates ubiquitination to a greater extent for the β-catenin peptide, our results suggest that eliminating interaction between the Rbx1 RING and the WHB is activating for multiple SCFs. Accordingly, WHB deletion mutants have also been found to stimulate SCF βTRCP/Cdc34-mediated polyubiquitination of IκB (ZQ. Pan, personal communication).

Figure 5
Functional analysis of mutations influencing RING conformational flexibility

Hindering RING orientational flexibility decreases CRL activity

Altered Rbx1 linker orientation in unmodified CRLs could allow juxtaposition of Ubc12’s Cys and the cullin NEDD8ylation site. Also, after cullin NEDD8ylation, varying RING positions could contribute multiple catalytic geometries for CRL-catalysis of polyubiquitination. Thus, based on modeling catalytic complexes, we designed deletions and insertions at Rbx1 Val38-Val39 to probe roles of linker positioning and flexibility. Deletion mutants test models for NEDD8ylation, as shortening the linker would not allow Rbx1’s RING to position Ubc12’s Cys at the NEDD8ylation site. By contrast, as there appears to be some slack in the linker in conformations modeled for ubiquitination, insertions of different sequences would test the role of linker positioning. Glycine insertions would accommodate rotation of the RING into a range of conformations. By contrast, extra valines, which have high propensity for adopting beta structures, would limit the RING to particular extended geometries.

We also designed mutants to probe conformations of individual linker residues. In both NEDD8ylated complexes, residues 36–38 extend similarly, with Φ angles ranging roughly from −60° to −90° and Ψ around +135° (Figure S8). By contrast, residues 39–41 display divergent orientations. Thus, Pro substitutions would test the importance of rotations observed about residues 39–41. Although prolines would be consistent with the structurally observed conformations of residues 36–38, if further rotations occur during catalysis a Pro substitution might be deleterious.

We first tested the effects of Rbx1 linker mutations on Cul1 NEDD8ylation (Figure 5A). Linker deletions (−1, −2) led to severe defects. Since Cul1 NEDD8ylation was not significantly affected by inserting residues into the Rbx1 linker (+1, +2, and +3), we deduce that the NEDD8ylation reaction establishes a minimum Rbx1 linker extension. Consistent with Rbx1 linker rotation, Pro substitutions for Ile37 and Val39 also impeded NEDD8ylation.

To test effects of mutations on CRL-mediated ubiquitination, we purified fully-NEDD8ylated forms for all the Cul1-Rbx1 mutants, except for the Val38-Val39 double deletion that could not be substantially NEDD8ylated even with attempts to force the reaction. Linker extension length plays an important role in SCF activity, as ubiquitination is substantially impaired with the Val insertion mutations (Figure 5C–F, S10, S11). However, there may be differences in geometries assumed by the different insertion mutants since there is not a linear correlation between the number of inserted residues and effects on monoubiquitination of the β-catenin peptide (Figure 5E–F). Also consistent with the crystal structures, Pro substitutions in place of Val39, Asp40, and Asn41, had deleterious effects on polyubiquitination, as did the Asp36 substitution (Figure 5C–D, S11). Only minor effects on monoubiquitin transfer were observed for these mutants, suggesting that conformational restriction imposed by Pro substitution primarily hinders polyubiquitin chain assembly (Figure 5E–F). The importance of linker flexibility is further substantiated by the roughly wild-type activity of linker mutants harboring multiple Gly insertions either before (B) or after (A) Val38-Val39, and also a single Gly inserted upstream of a deleterious Pro (Figure 5G–I).

Reoriented WHB~NEDD8 is poised to contact the ntd

Full-length NEDD8-modified CRL models were generated by superimposing NEDD8~Cul5ctd-Rbx1 and prior full-length cullin-Rbx1 structures, and aligned on the 4HB subdomain that contacts the ntd. In both NEDD8ylated CRL models, the WHB is positioned proximal to the ntd (Figure 2C, D), whereas the WHB is distal from the ntd in unmodified CRL structures (Figure 2B). Moreover, in the NEDD8ylated full-length CRL models, NEDD8, its linked WHB, and the 4HB together encircle ~3/4 of the cullin ntd (Figure 2C, D), with an important electrostatic network on NEDD8 (Wu et al., 2002) poised to contact the ntd. We speculate that subtle NEDD8-induced differences in the ctd-ntd interface may play a role in CRL function.

To test whether the WHB sequence also contributes to activation, we assayed activities for SCFs reconstituted with two Cul1/Cul5 chimeras which have homologous structures but different sequences in their predicted 4HB- and ntd- interaction surfaces (Figure 1, S1). The activities of un-NEDD8ylated chimeric complexes generally resembled their wild-type counterparts in three assays: (1) basal SCF ubiquitination (Figure 6A, B), (2) NEDD8ylation (Figure 6C, Figure S2), and (3) CAND1 inhibition of NEDD8ylation (Figure 6C). However, NEDD8-induced stimulation is substantially diminished for both chimeras (Figure 6B). Therefore, sequence-dependent interactions of the relocated WHB likely make important contributions to NEDD8-mediated activation. Additional studies are required to provide detailed insights into structural remodeling of a full-length NEDD8ylated CRL.

Figure 6
NEDD8-dependent role of the WHB sequence

Order of UBL targeting is an inherent CRL property, independent of E2 and UBL identity

Differences between the Culctd-Rbx1 and NEDD8~Culctd-Rbx1 structures raise the possibility that conformations may influence selection between cullin and substrate modification. This notion is consistent with results obtained while establishing our ubiquitination assays. In comparing wild-type unNEDD8ylated Cul1 and non-NEDD8ylatable Cul1Lys720Arg in SCF assays, we observe that (1) Cul1 itself is mono-ubiquitinated in a Lys720-dependent manner, (2) Cul1 mono-ubiquitination precedes ubiquitin transfer to phospho-p27 and β-catenin peptide, and (3) ubiquitin ligation to Cul1 activates SCFs. Consistent with our findings, in a study lacking NEDD8ylation components all active SCFs contained ubiquitin-modified Cul1 (Wu et al., 2003). Furthermore, in the absence of NEDD8, the yeast cullin Rtt101p is apparently modified by ubiquitin or another UBL (Laplaza et al., 2004). Although cullin NEDD8ylation is generally far more efficient than ubiquitination (not shown), our results demonstrate that irrespective of the UBL (NEDD8 or ubiquitin) or E2 (Ubc12 or UbcH5), cullin is the preferred first target of a single UBL. UBL ligation to Cul1 switches the target preference to substrate, and activates substrate polyubiquitination. We propose that the conformation of an unmodified CRL favors cullin as the first UBL target, and this modification repositions the cullin WHB and frees the Rbx1 RING to favor target polyubiquitination.

DISCUSSION

Hundreds of CRLs, generated by the combination of several cullin subunits with numerous substrate adaptors, catalyze a stunning array of reactions. (Cardozo and Pagano, 2004; Petroski and Deshaies, 2005; Tang et al., 2005). Studies of prototypic members of the SCF family indicate that each CRL may ubiquitinate tens of substrates, often on multiple acceptor lysines. Furthermore, SCFs containing structurally similar F-box protein substrate adaptors display substantial differences in their optimal degron-to-acceptor distances. These can vary by tens of residues even among substrates of a given SCF. Thus, the mechanistic requirements for CRL-mediated ubiquitination are complex.

Our data indicate that CRL conformational variability contributes to multiple catalytic functions (Figure 7B), including NEDD8ylation (Figure 7C), and target polyubiquitination (Figure 7D, 7E). The previously observed closed structures of unmodified 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 association with CAND1 (Goldenberg et al., 2004). Upon adaptor binding and CAND1 dissociation (Bornstein et al., 2006), possibly with influences from E2~UBL 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.

Figure 7
Conformational control of CRL activities

Interestingly, CRLs may display differences in intrinsic flexibility. A kink in Cul4A’s H29 helix subtly displaces the WHB and RING relative to other CRLs (Angers et al., 2006). Further, S. cerevisiae Cdc53p has a unique Gly-rich insertion immediately preceding the WHB (Figure S12), at the hinge we observe structurally, which may impart flexibility.

Despite conformational flexibility, the relative location of the RING is important. Insertion of a flexible hinge between Cul1’s ntd and ctd destroys ubiquitination (Zheng et al., 2002b), and the extension length of the linker tethering the RING is tightly constrained by a minimum required for Cul1 NEDD8ylation and a maximum limit for SCF-mediated polyubiquitination (Figure 5). In addition to the RING, the WHB is also an effector of NEDD8 activation, as evidenced by mutagenesis (Figure 6). Although further structural studies of full-length modified CRLs will be required to understand functions of the relocated WHB, we note that in the NEDD8~Cul5ctd-Rbx1 complexes, both the WHB and NEDD8 are poised to interact with the cullin ntd. We speculate that the WHB and/or NEDD8 may alter the conformation of the ctd-ntd junction in a way that contributes to ubiquitin E3 activity.

Many other aspects of CRL function, such as association with DCN1 during NEDD8ylation (Kurz et al., 2005), or the deNEDD8ylating COP9 Signalosome (Lyapina et al., 2001), could also be under conformational control. CRL activity may also be influenced by changes to substrate structures upon their phosphorylation, noncovalent association with partners, and/or ubiquitin ligation. Conformations associated with E2 “backside” binding to UBLs, and structural differences between polyubiquitin moieties dictated by linkage and chain length may also influence CRL activities (Brzovic et al., 2006; Pickart and Fushman, 2004; Sakata et al., 2007).

Conformational variation is emerging as a widespread theme in UBL conjugation cascades. For example, E1s and HECT E3s undergo domain rotations of greater than 100° that are important for catalysis (Huang et al., 2007; Huang et al., 2005; Verdecia et al., 2003). Also, analogous to “freeing” the Rbx1 RING, the Itch and Smurf2 E3s are activated by “freeing” their HECT domains from autoinhibitory intramolecular interactions (Gallagher et al., 2006; Wiesner et al., 2007). Similarly, the RING E3 Ubr1 is released from an autoinhibited state that prevents access to its substrate binding site (Du et al., 2002).

As revealed from structures of other signaling proteins composed of multiple subdomains, the modularity of enzymes present in UBL cascades probably amplifies their opportunity for conformational control. It seems likely that UBL attachment to other modular enzymes could drive reaction cascades by shifting conformational equilibria to disfavor inactive architectures and favor subdomain rearrangements that promote subsequent activities.

EXPERIMENTAL PROCEDURES

Constructs, Protein Preparation, and Antibodies

Human proteins were generated by standard molecular techniques, with construct coding sequences entirely verified. Ube1, UbcH5b, ubiquitin, APPBP1-UBA3, Ubc12, NEDD8, Skp1-Skp2, Skp1-βTRCP, CksHs1, CAND1, and CyclinA-cdk2 were prepared as described (Brown et al., 1999; Goldenberg et al., 2004; Huang et al., 2007; Huang et al., 2008; Schulman et al., 2000; Wu et al., 2003). Most assays used “split ‘n coexpress” Cul1-Rbx5–108 (and variants), shown previously to be structurally and biochemically identical to single polypeptide Cul1-Rbx1 (Zheng et al., 2002b). Ctds are Cul5401–780 or Cul1411–776, with solubilizing substitutions of ntd-binding 4HB hydrophobics (Cul5 - L407E, L439K, V440K; Cul1 L421E, V451E, V452K, Y455K) (Zheng et al., 2002b). Culctd-Rbx1s coexpressed as GST-Culctd-MBP-Rbx1 were purified by glutathione affinity. GSTCulctd-MBPRbxs were NEDD8ylated in 30mM Tris-Cl, 50mM NaCl, 10mM MgCl, 10mM ATP, pH 8.0 for 5 h at 4°C with 0.04x APPBP1-UBA3 and Ubc12, 2.5x NEDD8. After TEV protease treatment, Culctd-Rbxs were purified by cation exchange and gel filtration, concentrated to 10–15 mg/ml in 25mM Hepes or Tris-Cl, 150mM NaCl, 5mM DTT, pH 7.0 or 7.6, aliquotted, flash-frozen, and stored at −80°C. For crystallography, NEDD8 Leu62SeMet was used, as the additional Se facilitates structure determination without affecting function (D. Huang and BAS, unpublished).

For biochemistry, 8µM Cul1-Rbx1 (and variants) were NEDD8ylated in 50mM Tris, 150mM NaCl, 2.5mM MgCl2, 1.25mM ATP, pH 7.6 with 700nM APPBP1-UBA3, 1.8 µMUbc12, 60µM NEDD8 in 50mM Tris, 150mM NaCl, 2.5mM MgCl2, 1.25mM ATP, pH 7.6, for 30 min at rt (higher enzyme concentrations/longer reaction times for some variants). After adding DTT to 10mM, NEDD8ylated Cul-Rbxs were purified by gel filtration. GST-p27 (the version that can be phosphorylated on T187 by cdk2, (Vlach et al., 1997), also with the S10A mutation that restricts phosphorylation to T187(Hao et al., 2005)) was purified by glutathione affinity, treated with thrombin, and purified to homogeneity by gel filtration. Cdc34BKR (a Lys>Arg version that cannot be inhibited by autoubiquitination (Scaglione et al., 2007)) was purified similarly. 4µM p27 was phosphorylated on T187 at 30°C for 30min at 1:1 with CyclinA-cdk2 in 40mM Tris-HCl, 10mM MgCl2, 1mM ATP, 1mM DTT, pH 7.6. β-catenin phosphopeptide (Wu et al., 2003), has a C-terminal biotin attached via a PEG linker, which we found does not influence function (DCS and BAS, not shown). Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198). ΔWHB mutant terminates after Cul1 residue 692. The chimera junctions are at Cul1/Cul5 residues 697/701 (A) and 707/711 (B) (Figure S1).

Crystal structure determination

Cul5ctd-Rbx1 crystals were grown by microseeding 4°C hanging drops 1:1 with ~2% PEG3350, 0.1M HEPES, 0.2M L-Pro, pH 8.0, in P212121 with a=63.0, b=65.5, c=141.1 and one complex per au, and frozen in 5% PEG3350, 0.1M HEPES, 0.2M L-Pro, 8% glycerol, 8% ethylene glycol, 8% sucrose, pH 8.0. NEDD8~Cul5ctd-Rbx1 crystals were grown by microseeding 4°C hanging drops 1:1 with ~19% PEG3350, 275mM (NH4)2PO4, 5mM DTT, in P212121 with a=88.2, b=122.4, c=128.6 and two complexes per au, and frozen in 19% PEG3350, 250mM (NH4)2PO4, 10mM DTT, 8% glycerol, 8% ethylene glycol, 8% sucrose. Data were processed with HKL2000 (Otwinowski and Minor, 1997). SAD phases for NEDD8~Cul5ctd-Rbx1 were obtained with SOLVE with 24 of 36 Ses, with hand determination/density modification by RESOLVE (Terwilliger and Berendzen, 1999). Initial maps showed continuous density for NEDD8, Cul5ctd, and Rbx1 19–35. Rbx1’s RINGs were built by bootstrapping in O (Jones et al., 1991). Anomalous data collected at 1.283 Å showed zincs and confirmed RING positions. Cul5ctd-Rbx1 structure was determined by molecular replacement using PHASER (Storoni et al., 2004) with 4HB, α/β, and WHB subdomains from NEDD8~Cul5ctd-Rbx1. SeMet anomolous difference fourier maps from data collected at 0.98Å confirm SeMet and zinc positions. Models were refined with CNS (Brünger et al., 1998) and contain: Cul5ctd-Rbx1 – Cul5 401–517 and 520–780, and Rbx1 19–63 and 67–108; NEDD8~Cul5ctd-Rbx11 – all NEDD8 residues, Cul5 401–515 and 520–780, and Rbx1 20–105; NEDD8~Cul5ctd-Rbx12 – all NEDD8 residues, Cul5 401–517 and 520–780, and Rbx1 19–50 and 53–105, with 58–60, 63–65, 71, 78, 93–94 built into patchy density and thus assigned zero occupancy. Details are in Supplementary Table 1. We presume the Rfree for NEDD8~Cul5ctd-Rbx1 reflects high RING mobility.

SAXS

The pair-distribution function (P(r)), calculated with GNOM (Svergun, 1992), for Cul1ctd-Rbx1 was indicative of a globular particle whereas NEDD8~Cul1ctd-Rbx1 showed a shifted maximum and extended tail. The NEDD8~Cul1ctd-Rbx1 sample was properly folded as revealed by the Kratky plot (Kratky and Porod, 1949) indicating the extended nature of this sample with respect to Cul1ctd-Rbx1. Crystal structures of Cul1ctd-Rbx1 and NEDD8~Cul5ctd-Rbx1 were used in rigid body modelling applying molecular dynamics as described (Hammel et al., 2005; Hammel et al., 2007). Additional details are in Figure S7.

Biochemical assays

NEDD8ylation was assayed with 500nM APPBP1-UBA3, 1.2µM Ubc12, and 4µM Cul1-Rbx1 and variants, initiated with 60µM NEDD8 in 50mM Tris-HCl, 150mM NaCl, 2.5mM MgCl2, 1.25mM ATP, pH 7.6. 1:1 complexes with CAND1 were used in Figure 6. Reactions were stopped with SDS sample buffer, products separated by SDS-PAGE, and visualized by Coomassie or western. Polyubiquitination was assayed at rt with 150nM Ube1, UbcH5b (1µM in Figure 5 and Figure 7, and 4µM in Figure 5A and Figure 6), 75 or 200nM SCF (β-catenin or p27), and 10µM β-catenin phosphopeptide or 0.2µM phospho-p27-CyclinA-Cdk2 in 75mM Tris-HCl, 100mM NaCl, 1 mg/ml BSA, 10mM MgCl2, 1mM ATP, ATP regenerating system, pH 7.6, and started with 130µM His-Ub. As unmodified β-catenin phosphopeptide runs off gels, dot-blots estimate each SCFβTRCP polyubiquitinates ~30 β-cats/hr under these conditions. For pulse-chase assays, 8.3µM UbcH5b was loaded for 25min at rt with 230nM Ube1, 65µM Ub(K>R) in 100mM Hepes, 100mM NaCl, 1mg/ml BSA, 5mM MgCl2, 1mM ATP, pH 7.6, and quenched with 50mM EDTA for 5min on ice. 1.2µM UbcH5b~Ub(K>R) thioester conjugate was diluted into chase mixes of 0.5µM SCF, 0.5µM phospho-p27-CyclinA-Cdk2 or 5µM β-catenin phosphopeptide, 75mM Tris, 100mM NaCl, 50mM EDTA, 1mg/ml BSA, pH 7.6, at 4°C (β-cat) or rt (p27). Ub reactions were stopped with DTT buffer, separated by SDS-PAGE, and products detected by immunoblotting.

For disulfide crosslinking, Cys pair A is Cul1K759C-Rbx1A63C (Figure S1), in “split ‘n coexpress” Cul1-Rbx1 (Zheng et al., 2002b). An additional Cul1 C752A mutation (also in controls) minimized background crosslinking to this Cul1 Cys. Cys pair B is Cul1 with a C-terminal Gly-Cys extension (+2Cys) and Rbx1 L88C. Cys pair B and controls were expressed in insect cells as described (Zheng et al., 2002b) with Cul1 as a single polypeptide for immunodetection. Oxidation was for 10s at rt with 10µM NatT and ~15µM Cul1-Rbx1 and Cys variants, quenched for 5min with 20µM NEM. Disulfide crosslinked Cul1 and Rbx1 separated from non-crosslinked proteins by nonreducing SDS-PAGE (Figure 4) or after reduction by DTT (Figure S9).

Limited proteolysis was performed in PBS at rt with 7µM Cul1-Rbx1 or NEDD8~Cul1-Rbx1, and 21µM Endoproteinase Glu-C. Mass spectrometry and Edman degradation are consistent with initial cleavage at Cul1 Glu691, Glu695, and Glu697.

Supplementary Material

01

ACKNOWLEDGEMENTS

We are grateful to D. King and S. Zhou for mass spectrometry/Edman sequencing, J. Endicott for cdk2 plasmid, C. Ross and Staff at ALS 8.2.1, ALS 12.3.1, and NSLS X25 beamlines, V. Pagala, P. Murray, R. Deshaies, ZQ. Pan, J.W. Harper, M. Lee, B. Dye, D. Miller, S. Bozeman and members of the Schulman lab for assistance, advice, and/or discussions. ALS is supported U.S. DOE Contract No. DE-AC02-05CH11231, and NSLS by DE-AC02-98CH10886. This work was supported in part by American Lebanese Syrian Associated Charities of St. Jude, the Howard Hughes Medical Institute, a Beckman Young Investigator Award, a Pew Scholar Award, the Phillip and Elizabeth Gross Foundation, the NIH (R01GM069530), and DOD (DAMD17-03-0420) to BAS, and by an American Cancer Society fellowship to DMD. BAS is an Investigator of the Howard Hughes Medical Institute.

Footnotes

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ACCESSION CODES

Coordinates/structure factor amplitudes have Protein Data Bank have accession codes 3DPL and 3DQV.

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