Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

The E1 (activating), E2 (conjugating), and E3 (ligating) ubiquitination cascade plays a key role in controlling cellular protein levels by catalyzing the polyubiquitination of substrate proteins, leading to their subsequent proteasomal degradation.1−3 The E3 ubiquitin ligases impart specificity for this process and act by bringing a ubiquitin-loaded E2 enzyme and the substrate into close proximity to allow the ubiquitin transfer to occur.4 The Cullin-RING ligases (CRLs) function as multisubunit protein complexes (Figure ​(Figure1A).1A). They are the largest family of E3 ligases in eukaryotes, consisting of ∼400 members5 that target ∼20% of the proteins degraded by the proteasome.6 CRLs are therefore involved in a myriad of cellular processes, including cell-cycle control, gene transcription, and signal transduction,7 so it is of great interest to study how these complex molecular machines assemble and function. Figure 1 ASB9 is part of a Cullin-RING E3 ubiquitin ligase complex. (A) Cartoon showing the assembly of Cullin-RING E3 ubiquitin ligases. ASB9 acts as a substrate recognition subunit (SRS) utilizing Elongin B and Elongin C as adaptor proteins to bind the Cullin ... Cullin (Cul) proteins are composed of two domains. The N-terminal domain consists of three repeats of five α-helix bundles (Figure ​(Figure1B)1B) and binds to a substrate recognition subunit (SRS), usually via adaptor proteins, whereas the C-terminal domain binds to RING-box proteins (Rbx) to recruit the ubiquitin-loaded E2 enzyme (Figure ​(Figure11A).8 Therefore, Cullin acts as a scaffold on which to build the modular CRL functional complexes. There are seven Cullin proteins in the human genome (Cul1, Cul2, Cul3, Cul4a, Cul4b, Cul5, and Cul7),7,9 and each one binds to specific adaptor proteins and motifs within the SRS. Cul1 binds F-box proteins via the adaptor Skp1,10 Cul4 recruits WD40-containing proteins via linker-damaged DNA binding protein (DDB1),5 and Cul3 is unusual in that it recruits the adaptor and SRS in the same Bric-a-Brac, Tramtrack, and Broad complex (BTB)-domain proteins.11 In contrast, Cul2 and Cul5 both use Elongin B and Elongin C as adaptor proteins to bind VHL-box12 and SOCS-box proteins,13 respectively, as part of ECS-type (EloBC–Cullin–SOCS-box) CRLs. CRL ligase activities are controlled by several complex regulation mechanisms, including activation by conjugation with the protein NEDD8 (neural precursor cell expressed, developmentally downregulated 8) and inhibition by binding of CAND1 (cullin-associated and neddylation-dissociated 1).14 Interestingly, it is emerging that many CRLs may function as dimers as well as or instead of as monomeric multisubunit complexes.15−17 CRL dimerization interactions tend to occur through the adaptor proteins or SRS, for example, BTBSPOP–Cul3,18 BTBKLHL11–Cul3,19 and Fbx4–Skp1.20 The ankyrin-repeat and SOCS-box (ASB) proteins are postulated to act as substrate-recognition domains in ECS-type CRL complexes.21−24 However, this protein family is currently poorly understood, and there is limited knowledge of their degradation substrates and cellular roles. Crucially, no studies to date have investigated the assembly of ASB E3 ligase component structures or their interaction affinities. Here, we provide a biophysical and structural characterization of the noncovalent complex formed between ankyrin-repeat and SOCS-box protein 9 (ASB9), Elongin B (EloB), Elongin C (EloC), and Cul5. ASB9 (Figure ​(Figure1B,C)1B,C) has previously been shown to act as the substrate-recognition domain of an E3 ligase that includes EloB, EloC, Cul5, and Rbx2.25 ASB9 is predominantly expressed in the kidney and testes, and it has been shown to bind to and ubiquitinate brain-type cytosolic creatine kinase B25 and ubiquitous mitochondrial creatine kinase.26 However, the exact function of ASB9 remains unknown. Recent evidence suggests that ASB9 could be a biomarker for human breast cancer,27 and it has also been linked to colorectal cancer,28 suggesting that it may be a new potential drug target against these diseases. In this Article, we show that ASB9 forms a high-affinity 1:1:1:1 quaternary complex with EloB, EloC, and Cul5, and we describe a structural model for this complex that is consistent with ion-mobility mass spectrometry and temperature-dependent ITC data. These results present insights into the assembly of ECS-type CRLs and provide a platform for further investigation of the ASB protein family.