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15 a.a.
118 a.a.
96 a.a.
Key reference
DOI no: 10.1016/j.jmb.2008.06.038 J Mol Biol 381:928-940 (2008) PubMed id: 18590740
The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. J.J.Babon, J.K.Sabo, A.Soetopo, S.Yao, M.F.Bailey, J.G.Zhang, N.A.Nicola, R.S.Norton. ABSTRACT
Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.
Selected figure(s)
Figure 5.
Fig. 5. Tertiary structure of an SOCS3 SOCS box–elonginBC complex. (a) Ribbon diagram of the SOCS3 SOCS box (red) in complex with elonginC (blue) and elonginB (green). The interaction of the SOCS box occurs exclusively with elonginC and is mediated mostly by hydrophobic interactions. ElonginBC forms a tightly associated complex with the core of the association being a continuous β-sheet formed by residues from both proteins. A number of side-chain hydrophobic interactions further stabilise the complex. (b) Close view of the SOCS box elonginC interface with hydrophobic side chains from the SOCS box (red) labelled. The hydrophobic residues from elonginC (blue) that form the interface are shown in grey stick representation. (c) Ribbon diagram of the SOCS3 SOCS box in complex with elonginBC [same color scheme as in (a)] shown overlaid on the SOCS2/elonginBC structure (cyan).Figure 6.
Fig. 6. Key residues required for elonginBC binding. An Ala scan of the SOCS3 SOCS box to determine residues required for elonginBC binding was performed. Co-expression of 12 SOCS box domain constructs, each containing a single Ala mutation, with elonginBC was performed in E. coli, and glutathione Sepharose was used to pull down GST-labelled proteins present in the cell lysate. (a) The following mutations completely interfered with elonginBC binding: Val1, Thr3, Leu4, Leu7, Cys8, Arg9 and Val12. Of these, only the L4A mutation completely abolished binding. (c) Residues identified by Ala scan are highlighted on a surface representation of elonginBC where hydrophobic residues on the surface of elonginC are shown in yellow. The BC box of SOCS3 (red) is shown in cartoon representation with important side chains displayed in “stick” representation. ElonginC is shown in blue and elonginB in green.The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 381, 928-940) copyright 2008. Figures were selected by the author.
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