Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation

Abstract

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~Cul5(ctd)-Rbx1 and SAXS analysis of NEDD8~Cul1(ctd)-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.

Figure 1. Crystal structures of Cul5^ctd-Rbx1 and NEDD8~Cul5^ctd-Rbx1

A, Cul5^ctd(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 Cul5^ctd (green)-Rbx1 (navy), indicating positions of Helices 24 and 29 (H24, H29) and the 4HB-, α/β-, and WHB- subdomains. C, Cartoons of the two NEDD8 (yellow, gold)~Cul5^ctd (lime, sky)-Rbx1 (blue, violet) complexes in the asymmetric unit, with the Cul5 Lys724~NEDD8 isopeptide bond in sticks. D, Superposition of the 4HB-, α/β-, and WHB-subdomains for the two NEDD8~Cul5^ctd-Rbx1 complexes from the au, shown in three orientations, colored as in C. E, Superposition the 4HB-, α/β-subdomains from NEDD8~Cul5^ctd-Rbx1s and Cul5^ctd-Rbx1, oriented and colored as in B and C.

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

A, CAND1 (red)-Cul1 (ntd grey; 4HB, sky; α/β, lime; WHB, green)-Rbx1 (RING, blue) structure (Goldenberg et al., 2004), shown as a cartoon and semi-transparent surface, with the 4HB- and α/β-subdomains oriented as in the right view of 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, D, Models of full-length NEDD8~CRL structures generated by superimposing the α/β-and ntd-binding 4HB subdomains from NEDD8~Cul5^ctd-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 1C. NEDD8 mediated subdomain rearrangement eliminates the indicated CAND1 binding surface (red circle), and positions NEDD8 and its associated WHB to contact the cullin ntd (grey).

Figure 3. Small Angle X-Ray Scattering of NEDD8~Cul1^ctd-Rbx1 and Cul1^ctd-Rbx1

A, Pair-distribution (P(r)) function for NEDD8~Cul1^ctd-Rbx1 (blue) and Cul1^ctd -Rbx1 (black). Quality of fit (χ²) for individual open conformations sampled by molecular dynamics, and comparison of the experimental SAXS data (black) and theoretically-calculated SAXS-scattering profiles for the three best fitting conformations (red, blue, green) for Cul1^ctd-Rbx1 (closed – “C”, B) and NEDD8~Cul1^ctd-Rbx1 (open – “O”, C). Alternative closed conformations with flexibly linked NEDD8, sampled for data on NEDD8~Cul1^ctd-Rbx1, are shown in olive.

Figure 4. Probes of Conformational Change in Full-length NEDD8~Cul1^ctd-Rbx1

A, Cys pairs (A and B) at Cul1 and Rbx1 residues within 10 Å in the closed conformation, but widely separated in open conformations based on NEDD8~Cul5^ctd-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 or the indicated (A or B) Cys mutants of Cul1 and/or Rbx1. Cys As 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 producted detected by western blotting against Cul1 C-terminus (left), NEDD8 (middle), or Cul1 ntd (right).

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 (WT~N8), the non-NEDD8ylatable control (K720R), and the Cul1-Rbx1 WHB deletion mutant (ΔWHB). Left - SCF^βTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide, detected by western blotting with anti-biotin antisera. Right - SCF^Skp2/CksHs1-mediated polyubiquitination of phospho-p27, detected by western blotting with anti-p27 antisera. B, NEDD8ylation time-course for wild-type (wt) Cul1-Rbx1, non-NEDD8ylatable (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. C, SCF^βTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide or D, SCF^Skp2/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, SCF^βTRCP and a biotin-labeled β-catenin phosphopeptide and F, SCF^Skp2/CksHs1 and phospho-p27. G, NEDD8ylation for Rbx1 linker triple Val or Gly insertions (left), and indicated Pro mutants or those with upstream Gly insertions (^ ^G) (right). H, SCF^βTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide and I, SCF^Skp2/CksHs1-mediated polyubiquitination of phospho-p27 for fully NEDD8ylated SCFs harboring Rbx1 linker triple Val or Gly insertions (left), and indicated Pro mutants or those with upstream Gly insertions (^ ^G) (right).

Figure 6. NEDD8-dependent role of the WHB sequence

Time-courses of polyubiquitination by A, SCF^βTRCP and B, SCF^Skp2/CksHs1 reconstituted with fully-NEDD8ylated wild-type Cul1-Rbx1 (WT~N8) or Cul1/5 chimeras (A or B~N8), and their non-NEDD8ylatable counterparts (K>R), as in A. C, NEDD8ylation time-course and CAND1 inhibition for wild type Cul1-Rbx1 and the Cul1/5-Rbx1 chimeras. Reactions were separated by SDS-PAGE and visualized by Coomassie staining.

Figure 7. Conformational control of CRL activities

A, Polyubiquitination reactions with SCF^βTRCP/β-catenin phosphopeptide (left), and SCF^Skp2/CksHs1/ phospho-p27 (right) reconstituted with non-NEDD8ylatable (K720R), un-NEDD8ylated wild-type (WT), and fully NEDD8ylated Cul1-Rbx1 (WT~N8). Reaction products were detected by immunoblotting, top panels with anti-biotin (left) or anti-p27 (right), middle with anti-His (Ubiquitin; green * - Cul1~Ubiquitin), and lower with anti-Cul1 C-terminus antisera. B–E, Models of conformations involved in CRL activities. B, Structural model of SCF^βTRCP-β-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 with catalytic Cys a yellow sphere, Rbx1 in blue, the ctd portion of Cul1 in green, Cul1 ntd in grey, the substrate adaptor Skp1- F-box protein (Fbp) βTRCP complex in purple and magenta, and β–catenin peptide target in green sticks. Approximate distances to be spanned for NEDD8 transfer to the cullin target Lys (green stick) and for NEDD8 transfer 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 α/β-and ntd-binding 4HB subdomains from NEDD8~Cul5^ctd-Rbx1 and Cul1-Rbx1. The E2 Cys and peptide target 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 polyubiquitin chain extension.