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Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domain

SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase‐signal transduction and activator of transcription (JAK‐STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N‐terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C‐terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine‐inducible SH2‐containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C‐terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single α‐helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding.