Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands

Edmond M Linossi,Kunlun Li,Gianluca Veggiani,Cyrus Tan,Farhad Dehkhoda,Colin Hockings,Dale J Calleja,Narelle Keating,Rebecca Feltham,Andrew J Brooks,Shawn S Li,Sachdev S Sidhu,Jeffrey J Babon,Nadia J Kershaw,Sandra E Nicholson

doi:10.1038/s41467-021-26983-5

Abstract

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.

Highlights

Suppressor of cytokine signaling (SOCS)[2] protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade
The peptide library was cycled through five rounds of selection for binding to a fusion protein consisting of the SOCS2-Src homology 2 (SH2) domain and SOCS box fused to the C-terminus of glutathione S-transferase (GST), in complex with elongins B and C (GST-SOCS232–198-EloB/C; SOCS2 lacking the N-terminal region) (Fig. 1a)
To maximize the chances of isolating highly specific peptides, prior to binding to the GST-SOCS232–198-EloB/C complex, the library was incubated with bovine serum albumin (BSA) and GST to deplete the phage pool of non-specific peptides

Summary

Introduction

Suppressor of cytokine signaling (SOCS)[2] protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and enhances binding affinity. The binding specificities between different SH2 domains and the sequence surrounding the phosphotyrosine motif in individual proteins guarantee the accurate recognition of the target protein and tightly regulated signal transmission. The SH2 domain is a small protein module consisting of three β-strands flanked by two α-helixes These secondary structural elements and the loops that connect them form two pockets; one is the highly conserved phosphotyrosine binding pocket, and the second is a hydrophobic pocket that typically accommodates hydrophobic residues in the target protein located in the “+3” position following the phosphotyrosine[2,3]. This recruitment allows the ubiquitination and proteasomal degradation of targets bound to the SOCS-SH2 domains and classes the SOCS proteins as Cullin-RING E3 ligases (CRLs)[23]

Methods

Results

Conclusion