Suppressor of cytokine signalling 2 (SOCS2) is the classical negative regulator of growth hormone receptor (GHR) signalling. It contains a central SH2 domain that binds to phosphorylated tyrosine (pTyr) motifs on the cytoplasmic tail of GHR, while the conserved SOCS box domain recruits the Cullin5/Rbx2 RING-type E3 ubiquitin ligase complex via association with the adaptors Elongin B and C. This allows SOCS2 to target GHR for proteasomal degradation in a ubiquitin-dependent manner, thus preventing excessive GHR signalling. Previous studies have shown that the SH2 domain of SOCS2 is essential for its function. Genetic deletion of SOCS2 or mutations to the key residues of the SH2 domain involved in the pTyr interaction ultimately lead to gigantism in mice.
Although SH2 domains typically bind long, extended amino acid motifs, we found that the small molecule phosphomimetic, phenyl phosphate, is sufficient to engage the pTyr binding pocket (P0) of SOCS2, albeit with low affinity. NMR spectra of the SOCS2-SH2 domain with phenyl phosphate produced extensive chemical shift perturbations that were not isolated to the P0 pocket, while thermal shift assays confirmed ligand engagement by showing a 5°C increase in melting temperature.
We were able to recapitulate this increase in thermal stability in a ligand-independent manner by introducing negatively charged residues into the P0 pocket of SOCS2. We found that point mutations S76D and T83E result in a loss of binding to a phosphopeptide from GHR, as evidenced by thermal shift assays and solution NMR studies. Additionally, these mutations caused an increase in the overall thermal stability of the SH2 domain compared to the native sequence, suggesting that there is an intra-domain interaction that stabilises the overall domain architecture.
We postulate that the negatively charged residues introduced in these positions act as a phosphomimic, engaging the P0 pocket while excluding genuine pTyr ligands. We propose that these mutant variants can serve as good negative controls to investigate SOCS2 function, and that these types of mutations are potentially broadly applicable to other SH2 domain-containing proteins.