The specificity of protein-protein interactions is key to the successful execution of virtually every biological process. While some proteins have evolved to interact with pre-determined partners, other protein-protein interactions are driven by so-called modules that have evolved more generic recognition properties. Two such modules were recognised very early on by homology with the Src protein tyrosine kinase, namely SH2 domains (Sadowski et al, 1986) and SH3 domains (Mayer et al, 1988; Stahl et al, 1988; the SH1 domain is the protein kinase domain itself). SH3 domains bind to proline-rich peptides, while SH2 domains bind to phospho-tyrosines (pY). The reversible phosphorylation of tyrosine represents an on/off switch that in this case would directly regulate the binding between an SH2-domain protein and its target in each signal transduction pathway.
SH2 domains are approximately 100 amino acids long and contain both a fairly generic pY binding module and a neighbouring module conferring sequence specificity. Approximately 140 SH2 domains have been identified in 120 human proteins, and sequence analysis suggests that they have evolved from a recent common ancestor (Liu et al, 2006; Tinti et al, 2013).
Adhirons and Affimers are classes of non-Antibody Binding Protein that have been engineered to present constrained peptides as recognition surfaces. The scaffolds are small (~100 amino acids, 11 kDa, 3 nm in diameter), stable (Tm of 70oC to over 100oC), monomeric proteins that lack di-sulphide bonds, and are therefore uniquely suited to both a wide range of applications in protein detection (eg western blotting, enzyme-linked sorbent assays) and capture (affinity purification) and use inside human cells, to interfere with natural protein-protein interactions. Each scaffold is capable of displaying two peptides as loops of varying lengths. To create libraries of candidate binders we use codon-based oligonucleotide synthesis to control amino acid diversity so that 19 of the available natural amino acids are evenly represented in the library. The presented peptides are sufficiently close in space that they may interact, and thus the scaffold may present not just loops but also a variety of flat surfaces for interaction.
Working with Avacta Life Sciences we established a project with the primary objective to develop a tool box of reagents to target and block SH2 domain dependent protein interactions. Specifically binding reagents will be identified that bind to all of the SH2 domains and used in a range of assays and techniques to measure specificity and ability to inhibit SH2 domain function.