Engineering Src homology 2 domains for the study of protein tyrosine O-sulfation

Protein tyrosine O-sulfation is an essential posttranslational modification required for effective biological processes such as hemostasis, inflammatory response, and visual phototransduction. Due to its unstable nature under mass spectrometry conditions and residing on low abundance cell surface proteins, sulfated tyrosine (sulfotyrosine) residues are difficult to detect or analyze. Enrichment of sulfotyrosine-containing proteins (sulfoproteins) from complex biological samples are typically required before analysis. In this work, we seek to engineer the phosphotyrosine binding pocket of a Src Homology 2 (SH2) domain to act as an anti-sulfotyrosine antibody mimic. Using tailored selection schemes, several SH2 mutants are identified with high affinity and specificity to sulfotyrosine. Further molecular docking simulations highlight potential mechanisms supporting observed characteristics of these SH2 mutants. Utilities of the evolved SH2 mutants were demonstrated by the detection and enrichment of sulfoproteins.