Multimeric Complexes among Ankyrin-Repeat and SOCS-boxProtein 9 (ASB9) ElonginBC and Cullin 5: Insights into the Structureand Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

摘要

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 highaffinity to the Cullin 5 N-terminal domain (Cul5NTD) butnot to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling andis consistent with IM–MS and temperature-dependent ITC data.This is the first experimental study to validate structural informationfor the assembly of the quaternary N-terminal region of an ASB CRLcomplex. The results suggest that ASB E3 ligase complexes functionand assemble in an analogous manner to that of other CRL systems andprovide a platform for further molecular investigation of this importantprotein family. The data reported here will also be of use for thefuture development of chemical probes to examine the biological functionand modulation of other ECS-type CRL systems.