Structural assembly of cullin-RING ubiquitin ligase complexes

Abstract

The cullin-RING ubiquitin ligases (CRLs) are the largest family of multi-subunit E3 ligases in eukaryotes, which ubiquitinate protein substrates in numerous cellular pathways. CRLs share a common arched scaffold and a RING domain catalytic subunit, but use different adaptors and substrate receptors to assemble unique E3 machineries. In comparison to the first CRL structure, recent findings have revealed increased complexity in the overall architecture and assembly mode of CRLs, including multi-domain organization, inter-domain flexibility, and subunit dimerization. These features highlight the capacity of CRLs to catalyze protein ubiquitination under distinct cellular contexts and in response to diverse signals. As the first installment of a two-review series, this article will focus on recent advances in our understanding of CRL assembly mechanisms.

Figure 1

Modular assembly of cullin-RING ubiquitin ligases (CRLs). Cullin scaffold proteins (Cul1-5, green) in complex with Rbx1/2 (R, red) form the catalytic cores of CRLs. Ubiquitin (U)-charged E2 enzymes are recruited to CRLs by Rbx1/2. Skp1, Elongin C, and DDB1 serve as the adaptor (light blue) of CRL1(SCF), CRL2/5, and CLR4, respectively. F-box proteins, BC-box proteins, BTB-domain proteins, and DCAF proteins function as the substrate receptors (magenta) of CLR1(SCF), CRL2/5, CLR3, and CLR4, respectively. Adaptor binding regions of all cullins are colored in cyan.

Figure 2

Structural models of CRL1 and CRL2 showing a common assembly mode. (a) Structural model of SCF^Skp2 obtained by superimposing the Skp1-Skp2 complex and the Cul1-Rbx1-Skp1-Skp2 F-box domain structure [8,9]. The E2 (orange) is modeled by superimposing Rbx1 RING domain with the UbcH7-bound c-Cbl RING domain [12]. Zinc atoms are shown as yellow spheres. The two Cul1 α-helices used to bind Skp1 and Skp2 F-box motif are colored in cyan. (b) Structural model of SCF^β-TrCP in complex with phosphorylated β-catenin degron peptide. The model is obtained by superimposing Skp1 in the structures of Skp1–β-TrCP and Cul1-Rbx1-Skp1-Skp2 F-box domain complexes [63]. β-TrCP uses the top surface of its WD40-repeat β-propeller domain to recognize and present its substrate β-cateinin. (c) Structural model of CRL2^VHL obtained by superimposing Elongin C in the Elongin BC-VHL complex and Skp1 in the Cul1-Rbx1-Skp1-Skp2 F-box domain complex [17]. (d) Structural model of CRL5^SOCS2 obtained by superimposing Elongin C in the Elogin BC-SOCS2 complex and Skp1 in the Cul1-Rbx1-Skp1-Skp2 F-box domain complex [18].

Figure 3

Crystal structures of CRL4^SV5-V and DDB1 in complex with viral hijackers, DCAF H-box motif, and DDB2. (a) Crystal structure of the Cul4A-Rbx1-DDB1-SV5-V complex with all subunits in (near) full-length forms [19]. The two α-helices used by Cul4A to bind DDB1 are colored in cyan. The BPB domain of DDB1 is colored (slate) differently from the BPA-BPC double propeller (marine) for clarity. (b) Bipartite interaction of SV5-V with DDB1 [22]. The SV5-V N-terminal α-helix is inserted into the pocket created in between the DDB1 BPA and BPC β-propeller domains. The SV5-V C-terminal domain containing two zinc ions (yellow) forms an additional interface with the DDB1 BPC domain. (c) Superimposed crystal structures of DDB1 in complex with the H-box motif of either HBx (magenta) or DCAF9 (orange) [27•]. Superimposable structures of DDB1 bound to the H-box motif of other DCAFs are not shown. (d) Crystal structure of DDB1 in complex with the DCAF protein DDB2 and UV-damaged DNA duplex [28••]. DDB2 binds DDB1 via a bipartite interface, similar to SV5-V. The N-terminal α-helix of DDB2 sits at the same position as that of SV5-V and the H-box motif of HBx and other DCAFs. DDB2 uses its narrower top surface, which faces away from DDB1, to recognize DNA lesions. Note that the position and orientation of the DDB1 BPB domain relative to the BPA-BPC double propeller is different in all structures.

Figure 4

Dimerization of CRL substrate receptors. (a) A schematic model showing that the asymmetric SPOP dimer recruits two copies of Cul3-Rbx1 and can bind a single substrate polypeptide by recognizing two degrons via two MATH domains [41••]. Degrons are represented by the rounded ends of substrate. (b) Crystal structure of the asymmetric SPOP dimer [41••]. Each MATH domain in SPOP is in complex with a Puc degron peptide. The BTB domains of SPOP mediate dimer formation, while the 3 box domains promote Cul3 binding. (c) Crystal structures of Keap1 Kelch β-propeller in complexes with two distinct degrons of Nrf2 [32,33,54]. A schematic drawing of the full-length Keap1 dimer is shown at the bottom based on the two-site recognition model and EM reconstruction results. Each globular lobe of the Keap1 dimer contains a Kelch β-propeller and the IVR region. (d) Crystal structure of the dimeric Cdc4 D-domain and a top view of a schematic model of a dimeric SCF^Cdc4 proposed in [62]. The WD40-repeats domains of the Cdc4 dimer are represented by two circles.