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

Abstract

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma

“…For instance, CUL5, RBX2, Elongin B (TCEB2) and Elongin C (TCEB1) are part of the Cullin-ring complex that interacts with the SOCS box (Fig. 2), while polyubiquitin C (UBC) is a source of ubiquitin (Thomas et al, 2013). Another ASB10-interacting protein was identified as HSPA1A, which is also known as HSP72 or HSP70.…”

Insights into Cullin-RING E3 Ubiquitin Ligase Recruitment: Structure of the VHL-EloBC-Cul2 Complex

“…The cullin box consists of the remaining helix-turn-helix motif and interacts with cullins. The structures of various SOCS-box containing proteins (ASB9, SOCS2, SOCS3, SOCS4, GUSTAVUS, VHL, and Vif) bound to EloBC show that SOCS boxes are in similar conformations and use a conserved mechanism to bind EloBC (Babon et al, 2008; Bullock et al, 2006; Bullock et al, 2007; Guo et al, 2014; Kim et al, 2013; Muniz et al, 2013; Stanley et al, 2008; Thomas et al, 2013; Woo et al, 2006). However, different SOCS box proteins interact with different cullins.…”

Model of the Ankyrin and SOCS Box Protein, ASB9, E3 Ligase Reveals a Mechanism for Dynamic Ubiquitin Transfer

“…The CK Cullin-5 E3 Ubiquitin Ligase complex was built was previously described (Thomas et al 2013), with the ASB9(19-252)-CK complex of the equilibrated ZDOCK-1 pose, the equilibrated ZDOCK-2 pose, and cluster centroids from both simulations. The various E3 Ligases were uploaded to the AD-ENM (Elastic Network Model) server (Zheng & Brooks 2005).…”

“…Previous models of CRLs show a the distance of over 60 Å between ubiquitin, positioned on the C-terminal end of Cullin protein, and the target protein, positioned at the N-terminal end the Cullin. Thus, substantial conformational changes must be required for ubiquitin transfer (Thomas et al 2013; Duda et al 2008). …”

“…As one of eighteen members of the ASB family, the largest family of SOCS box containing proteins (Kohroki et al 2005), ASB9 is composed of an intrinsically disordered N-terminus, an ankyrin repeat domain and the SOCS box domain. While the SOCS box interacts with Elongin B, Elongin C, and Cullin 5 in the E3 ligase complex (Thomas et al 2013; Muniz et al 2013), the ankyrin repeat domain and N-terminal disordered segment interact with creatine kinase (Fei et al 2012; Balasubramaniam et al 2015). Creatine kinases play a critical role in the buffering of ATP/ADP ratios in excitable cells such as in cardiomyocytes and neural cells, and as such are crucial for cellular homeostasis in the vertebrate heart and brain (Wang et al 2011; Bush et al 2011).…”