Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

A novel exosite peptide enhances binding of the SOCS2-SH2 domain to phosphorylated targets (#256)

Edmond Linossi 1 2 , Dale Calleja 1 3 , Gianluca Veggiani 4 , Nadia Kershaw 1 3 , Shawn Li 5 , Jeffrey Babon 1 3 , Sachdev Sidhu 4 , Sandra Nicholson 1 3
  1. University of Melbourne, Parkville, Vic, Australia
  2. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  3. The Walter & Eliza Hall Institute, Parkville, VIC, Australia
  4. The Donnelly Centre, University of Toronto, Toronto
  5. Department of Biochemistry and Siebens-Drake Medical Research Institute, University of Western Ontario, London

Src Homology 2 (SH2) domains bind to linear tyrosine-phosphorylated motifs and play a crucial role in bridging upstream tyrosine kinases with downstream signalling pathways. SH2 domains typically bind their cognate phospho-sequences with dissociation constants ranging from 0.1-10 µM. The Suppressor of Cytokine Signalling proteins (CIS; SOCS1-7) are important feedback inhibitors of cytokine and growth factor signalling and act as adaptors for an E3 ubiquitin ligase complex, binding to target proteins through a central SH2 domain. In particular, SOCS2 is a negative regulator of growth hormone signalling and more recently has been shown to act as an immune checkpoint in dendritic cells, dampening anti-tumour immunity (Nirschl et al., Cell 2017). We have utilised phage-display technology to explore alternative binding surfaces on the SH2 domain of the SOCS family of proteins. These analyses identified a group of non-phosphorylated peptides that bound the SOCS2-SH2 domain with low micromolar affinity (Kd 1.5-5.0 µM), equivalent to that of the known SOCS2 site in the growth hormone receptor (GHR; pY595; Kd 1.0 µM). These peptides were non-competitive for phosphopeptide binding and surprisingly, enhanced SOCS2 affinity for the GHR pY595 peptide 20-fold (Kd 0.05 µM). Ongoing biochemical and structural studies aim to identify the binding site of these exosite peptides on SOCS2 and characterise the functional impact of enhanced SOCS2 affinity for the GHR and other phosphorylated targets in cells. Parallel studies will investigate SOCS2 interacting partners using recombinant protein pull-down/mass spectrometry in the presence and absence of these peptides. We hypothesis that additional proteins, which share sequence homology with these novel exosite peptides may regulate SOCS2 SH2 domain binding, providing an unanticipated level of regulation to SOCS protein function.