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Biophysical Studies on Interactions and Assembly of Full-size E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (S... - F1000Prime Article Recommendations
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Biophysical Studies on Interactions and Assembly of Fullsize E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (SOCS2)-ELONGIN BC-CULLIN 5-RING BOX PROTEIN 2 (RBX2).
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Bulatov E 1, Martin EM 2, Chatterjee S 2, Knebel A 3, Shimamura S 4, Konijnenberg A 2, Johnson C 3, Zinn N 4, Grandi P 4, Sobott F 2, Ciulli A 5 show author affiliations
J Biol Chem. 2015 Feb 13; 290(7):417891
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Pengbo Zhou F1000 Structural Biology Weill Medical College of Cornell University, New York, NY, USA.
Jeffrey Hannah F1000 Structural Biology Weill Medical College of Cornell University, New York, NY, USA.
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DOI: 10.3410/f.725275864.793504890 In this report, the authors utilize a variety of biophysical methods to reveal information on the structure, assembly, and potential activity of the SOCS2EloBCCul5Rbx2 (CRL5(SOCS2)) complex. Their work provides researchers with numerous alternative approaches for studying protein complexes particularly, those that cannot be crystallized as whole, intact structures. Cullin RING E3 ubiquitin ligases (CRLs) are protein complexes that consist of cullins (which provide a scaffold for the entire complex), E2 conjugating enzymerecruiting RING domain proteins (such as Rbx1 and Rbx2), substrate receptor proteins, and depending on the CRL an adaptor protein which acts as a bridge between cullins and various substrate receptors. The activity of CRLs is also regulated by the covalent attachment of an ubiquitinlike protein known as NEDD8, which is thought to induce conformational changes and facilitate ubiquitin transfer from the E2 enzyme to the substrate. Due to their roles in numerous cell processes and human diseases, researchers wish to understand more about the structure and function of CRLs, which may prove to be valuable therapeutic targets in certain contexts. Because of their modular nature, CRLs can be difficult to crystallize, which limits our understanding of their complete structures as assembled complexes. Bulatov et al. demonstrate that biophysical methods can be used to learn more about the structure and composition of the CRL5(SOCS2) complex and, presumably, other CRLs and protein complexes. SOCS2, the substrate receptor of the CRL5(SOCS2) complex, specifically binds to Tyrosine 595phosphorylated growth hormone receptor (GHR) but not its unmodified form. In order to study the active complex as it occurs in vivo, the authors used a beadconjugated phosphopeptide (GHR_pY595) to purify the ligase complex. As a control, the authors also used an unmodified peptide (GHR_Y595) to confirm its specificity. Despite the presence of a few other proteins in the precipitate (mostly members of the SCF E3 ligase complex), they found that GHR_pY595 was able to consistently bring down CRL5(SOCS2) but not GHR_Y595. Using isothermal titration calorimetry (ITC), they analyzed the affinity of SOCS2Elongin B/C (SOCS2EloBC) for both the GHR peptides and the Nterminal region of Cul5 (Cul5NTD). (The Elongin proteins act as a substrate adaptor, recruiting SOCS2 and other proteins to the Nterminus of Cul5.) According to their data, SOCS2EloBC has a much higher affinity for Cul5CTD than GHR_pY595. (Note: this is only a peptide and not the fulllength substrate.) They then examined the nature of the interaction between SOCS2EloBC and Cul5NTD by using available structural information and found that the majority of the interphase is hydrophobic and that the observed change in heat capacity for their ITC experiments falls in line with previous theoretical calculations from other groups. Some E3 ligases, such as CUL3, appear to dimerize in the cell and potentially demonstrate higher activity in this state. To determine how CRL5(SOCS2) may assemble in vivo, the authors performed sizeexclusion chromatography and multiangle light scattering (SECMALS) and native mass spectrometry (MS) techniques. According the ion mobility drift, mass spectra, and collision cross section (CCS) data, SOCS2EloBC, Cul5Rbx2, and SOCS2EloBCCul5Rbx2 display patterns that confirm that the complex assembles as a monomer. Furthermore, the experimental CCS values align very well with calculated (theoretical) values. To answer the question of how neddylation (conjugation of NEDD8 protein) affects the structure, they isolated complex subunits and performed in vitro neddylation reactions. After conducting native traveling wave ion mobilityMS (TWIMMS) analysis, they saw that neddylation did not appear to induce significant conformational changes to Cul5Rbx2 or the fully assembled complex. The change in CCS values following neddylation also suggests an ‘addon’ effect in which NEDD8 conjugation primarily appears to enlarge the complex without changing its structure. Unfortunately, the calculated difference in CCS based on theoretical models is too small to accurately determine whether a conformational change actually occurs. In summary, Bulatov et al. provide new insight on the CRL5(SOCS2) E3 ubiquitin ligase complex and offer alternative and supporting methods to crystallographybased structural analysis of CRLs and other protein complexes. Disclosures None declared Add a comment
Abstract: ABSTRACT
The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target posttranslationally 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, SOCS2EloBCCul5Rbx2 (CRL5(SOCS2)), binds more » 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
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Biophysical Studies on Interactions and Assembly of Full-size E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (S... - F1000Prime
phosphorylated growth hormone receptor peptides. Subsequently, SOCS2EloBC and fulllength Cul5Rbx2, 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 proteinprotein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for fullsize neddylated and unneddylated CRL5(SOCS2) complexes is supported by traveling wave ion mobility mass spectrometry data. RECOMMENDATIONS 1 | ABSTRACT | COMMENTS
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. DOI: 10.1074/jbc.M114.616664 PMID: 25505247
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Abstract courtesy of PubMed: A service of the National Library of Medicine and the National Institutes of Health.
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F1000 Faculty Reviews (incorporating F1000Prime Reports) are comprehensive, open access, topical reviews written by members of the prestigious F1000 Faculty. These peer reviewed articles provide context on emerging themes in biology and medicine. view all Cell Biology | Chemical Biology | Biochemistry | Physiology | Structural Biology | Biotechnology | Pharmacology & Drug Discovery
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Full text | PDF | Abstract on PubMed
Full text | PDF | Abstract on PubMed
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