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.
Highlights
The E1, E2, and E3 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]
To monitor the formation of the ternary complex among ASB9 and Elongin B−Elongin C (EloBC), the three proteins were coexpressed in Escherichia coli, and the complex purified using Ni-affinity chromatography and size-exclusion gel filtration followed by analysis using native nanoelectrospray ionization mass spectrometry
ASB9−EloBC was examined by mass spectrometry at three concentrations (20, 80, and 130 μM), all of which supported the existence of the ternary protein complex
Summary
The Cullin 5 N-terminal domain (Cul5NTD, residues 1−386) was expressed in E. coli, purified by Ni-affinity chromatography and size-exclusion gel filtration, and examined by nanoESI−MS. Cul5NTD (expected molecular weight from protein sequences, 45 690 Da). Cul5NTD eluted at a smaller elution volume during size-exclusion chromatography (54 mL) than ASB9−EloBC (57 mL) despite having a lower molecular weight (Figure 2), which is likely a result of its elongated shape (Figure 1B), causing it to behave as a larger protein by gel filtration. Monomeric Cul5NTD appears in the mass spectrum in the range 2800−4000 m/z with four charge states (12+ to 15+). As in the case of ASB9−EloBC, the monomer−dimer distribution could not be fitted to a binding isotherm and dimerization was not observed by size-exclusion chromatography, suggesting that. Cul5NTD is predominantly monomeric at physiological pH.
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