Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex

Abstract

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase

“…Most ubiquitination complexes require an E2 conjugating protein to provide the Ub moiety for the E3 ligases or multisubunit E3 ligase complexes (Freemont, 2000;Joazeiro and AM Weissman, 2000;Thrower et al, 2000;Zheng et al, 2002). We found that Smurf2 E3 ligase interacts with RNF11 through its WW domains (Figures 2 and 3).…”

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

“…The complex consists of Cul1's Nand C-terminal domains associated non-covalently with each other and with Rbx1. The split Cul1-Rbx1 complex was shown previously to possess the same 3-dimensional structure and ubiquitin ligase activity as the Rbx1 complex with Cul1 expressed as a single polypeptide in insect cells 45 . The 26-residue peptide corresponding to Ubc12's N-terminus, termed Ubc12N26 (sequence MIKLFSLKQQKKEEESAGGTKGSSKK), the five different selenomethionine-substituted Ubc12N26 peptides, and the ScrambledN26 peptide (sequence MKFQLKEIEAGKSKLKSGTSEKGQKS) were chemically synthesized with C-terminal amide blocking groups.…”

“…A mutant form of APPBP1-UBA3 lacking APPBP1's 254-258 loop and UBA3's N-terminal 11 residues, with UBA3's catalytic Cys216 mutated to Ala was used for crystallization 26 . Biochemical studies used full-length, wild-type E1s 44,45,28 or APPBP1-UBA3 mutants expressed and purified as for the wild-type E1; wild-type or mutant versions of Ubc12 28 as indicated; and the truncated active versions of ubiquitin and the UBLs human NEDD8 and S. cerevisiae Sumo (Smt3p) terminating with the sequence Gly-Gly44 , 26 , 28. The human Rbx1-Cul1 (split) complex was obtained in soluble form from E. coli by simultaneously coexpressing Rbx1, Cul1's N-terminal domain (residues 1-411), and Cul1's C-terminal domain (residues 411-776 at the C-terminus).…”

Ubiquitination-mediated protein degradation and modification: an emerging theme in plant-microbe interactions

“…The SCF-type E3 ligase is typically composed of four different polypeptides: SKP1 that serves as an adaptor protein; Cullin1 (CDC53); RING-finger protein RBX1 (or ROC1 and HRT1) that interacts with the Cullin1 and E2-conjugating enzyme; and an F-box protein that is responsible for recruiting substrates. Structural analysis indicated that Cullin1 (most of the time abbreviated as CUL1) acts as a scaffold that directs other members of the SCF complex to bring the ubiquitin-loaded E2s and the substrate in proximity to promote the transferring of ubiquitin directly from the E2 to the substrates [26]. Besides Cullin1, recent data indicated that Cullin2 and Cullin3 also form similar groups of E3 ligases, in which an elongin C-SOCS module or a BTB protein replaces the SKP1-F-box protein module npg in the Cullin1-type SCF E3 ligase, respectively [27][28][29].…”