PDBsum entry 3dpl

Ligase

PDB id: 3dpl

Contents

Protein chains

378 a.a. *

87 a.a. *

Metals

_ZN ×3

Waters ×73

* Residue conservation analysis

PDB id:

3dpl

Name:

Ligase

Title:

Structural insights into nedd8 activation of cullin-ring ligases: conformational control of conjugation.

Structure:

Cullin-5. Chain: c. Fragment: residues 401-780. Synonym: cul-5, vasopressin-activated calcium-mobilizing receptor, vacm-1. Engineered: yes. Mutation: yes. Ring-box protein 1. Chain: r.

Source:

Homo sapiens. Organism_taxid: 9606. Gene: cul5, vacm1. Expressed in: escherichia coli. Gene: rbx1, rnf75, roc1.

Resolution:

2.60Å R-factor: 0.244 R-free: 0.277

Authors:

D.M.Duda,B.A.Schulman

Key ref:

D.M.Duda et al. (2008). Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell, 134, 995-1006. PubMed id: 18805092 DOI: 10.1016/j.cell.2008.07.022

Date:

08-Jul-08 Release date: 30-Sep-08

Protein chain

Q93034  (CUL5_HUMAN) -  Cullin-5 from Homo sapiens

Seq: 780 a.a.

Struc: 378 a.a.*

Protein chain

P62877  (RBX1_HUMAN) -  E3 ubiquitin-protein ligase RBX1 from Homo sapiens

Seq: 108 a.a.

Struc: 87 a.a.

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class 2: Chain R: E.C.2.3.2.27 - RING-type E3 ubiquitin transferase.
• Reaction: S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + N₆- ubiquitinyl-[acceptor protein]-L-lysine
• Enzyme class 3: Chain R: E.C.2.3.2.32 - cullin-RING-type E3 NEDD8 transferase.
• Reaction: S-[NEDD8-protein]-yl-[E2 NEDD8-conjugating enzyme]-L-cysteine + [cullin]- L-lysine = [E2 NEDD8-conjugating enzyme]-L-cysteine + N₆-[NEDD8- protein]-yl-[cullin]-L-lysine

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1016/j.cell.2008.07.022

Cell 134:995-1006 (2008)

PubMed id: 18805092

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

D.M.Duda, L.A.Borg, D.C.Scott, H.W.Hunt, M.Hammel, B.A.Schulman.

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.

Selected figure(s)

Figure 1.
Figure 1. Crystal Structures of Cul5^ctd-Rbx1 and NEDD8 vert, similar 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] and [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) vert, similar Cul5^ctd (lime, sky)-Rbx1 (blue, violet) complexes in the asymmetric unit, with the Cul5 Lys724 vert, similar NEDD8 isopeptide bond in sticks.
(D) Superposition of the 4HB, α/β, and WHB subdomains for the two NEDD8 vert, similar Cul5^ctd-Rbx1 complexes from the au, shown in three orientations, colored as in (C).
(E) Superposition of the 4HB and α/β subdomains from NEDD8 vert, similar Cul5^ctd-Rbx1s and Cul5^ctd-Rbx1, oriented and colored as in (B) and (C).

Figure 4.
Figure 4. Probes of Conformational Change in Full-length NEDD8 vert, similar Cul1-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 vert, similar 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 (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).

The above figures are reprinted from an Open Access publication published by Cell Press: Cell (2008, 134, 995-1006) copyright 2008.

Figures were selected by an automated process.