Binding Specificity of Chimeric SH2 Domains

Peptide-binding domains are instrumental in cellular processes. Although these protein domains are non-catalytic, they act as modulators and scaffolds in cellular signaling and trafficking pathways. The SH2 domain is an important peptide-binding domain typically found in tyrosine kinase signaling pathways, which are important regulators of cell growth and division, amongst other cellular events. Mutations in these domains (tyrosine kinases and SH2s) can lead to human disease, particularly cancer. There are over 100 SH2 domains in the human proteome and they bind to short amino acid sequences that contain a phosphorylated tyrosine residue. Considering the overlapping specificities of SH2 domains, our lab is interested in how target recognition is encoded in this domain family and the effects on the cell. Previously, two conserved structural loops were identified as factors for determining the recognition selectivity of an SH2 domain. In our lab, we created several chimeric proteins, with the amino acid sequences of these loops in the Src SH2 domain exchanged for those from SH2 domains involved in other cellular pathways (e.g., PLCγ, Grb2, STAT1, etc.). We used biochemical techniques to characterize these chimeric domains with a high affinity peptide for the Src SH2 domain, revealing a wide variety of binding affinities, consistent with the critical role these loops play in target recognition. Here, we describe the details of this ongoing project and discuss our future directions into high throughput assays using these chimeras to further understand SH2 domain selectivity.