Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2) [Protein Structure and Folding]

December 11th, 2014 by Bulatov, E., Martin, E. M., Chatterjee, S., Knebel, A., Shimamura, S., Konijnenberg, A., Johnson, C., Zinn, N., Grandi, P., Sobott, F., Ciulli, A.

The multi-subunit Cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes and cancer, and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, amongst their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) binds phosphorylated growth hormone receptor (GHR) as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated GHR peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in E. coli and in Sf21 insect cells, respectively, were used to reconstitute CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike many other E3 ligases, the CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multi-angle static light scattering (SEC-MALS) and native mass spectrometry (MS). Affinities of the protein-protein interactions within the multi-subunit complex were measured by isothermal titration calorimetry (ITC). A structural model for the full-size CRL5SOCS2 is supported by travelling wave ion-mobility mass spectrometry (TWIM-MS) data.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2) [Protein Structure and Folding]