title: Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2).
abstract: The multisubunit 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, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5(SOCS2)), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5(SOCS2) can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5(SOCS2) complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5(SOCS2) was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5(SOCS2) complexes is supported by traveling wave ion mobility mass spectrometry data.
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, College of Life Sciences, and the Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
authors.forename: Emil
authors.initials: E
authors.lastname: Bulatov
authors.affiliation: the Department of Chemistry, University of Antwerp, 2020 Antwerp, Belgium, and.
authors.forename: Esther M
authors.initials: EM
authors.lastname: Martin
authors.affiliation: the Department of Chemistry, University of Antwerp, 2020 Antwerp, Belgium, and.
authors.forename: Sneha
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: the Medical Research Council Phosphorylation and Ubiquitylation Unit, College of Life Sciences, Sir James Black Center, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Satoko
authors.initials: S
authors.lastname: Shimamura
authors.affiliation: the Department of Chemistry, University of Antwerp, 2020 Antwerp, Belgium, and.
authors.forename: Albert
authors.initials: A
authors.lastname: Konijnenberg
authors.affiliation: the Medical Research Council Phosphorylation and Ubiquitylation Unit, College of Life Sciences, Sir James Black Center, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Clare
authors.initials: C
authors.lastname: Johnson
authors.affiliation: Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Zinn
authors.affiliation: Cellzome GmbH, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Paola
authors.initials: P
authors.lastname: Grandi
authors.affiliation: the Department of Chemistry, University of Antwerp, 2020 Antwerp, Belgium, and.
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, College of Life Sciences, and the Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom, a.ciulli@dundee.ac.uk.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ciulli
mesh_terms: D044842:Cullin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000075065:Elongin; D006801:Humans; D020014:K562 Cells; D013058:Mass Spectrometry; D008958:Models, Molecular; D000075687:NEDD8 Protein; D011485:Protein Binding; D011487:Protein Conformation; D050826:Suppressor of Cytokine Signaling Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C496871:CUL5 protein, human; D044842:Cullin Proteins; D000075065:Elongin; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C106643:SOCS2 protein, human; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014452:Ubiquitins; C118307:RNF7 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Biophysics; Cullin; E3 Ubiquitin Ligase; Isothermal Titration Calorimetry (ITC); Mass Spectrometry (MS); Post-translational Modification (PTM); Protein Assembly; Protein Complex; Protein-Protein Interaction