Engineering of a Small Protein Scaffold To Recognize Sulfotyrosine with High Specificity

Protein tyrosine O-sulfation is an essential post-translational modification required for effective biological processes such as hemostasis, inflammatory response, and visual phototransduction. Because of 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 antisulfotyrosine 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.

Automated summary

Protein tyrosine O-sulfation is crucial for various biological processes, but sulfated tyrosine residues are challenging to detect due to their instability and low abundance. This study engineered SH2 mutants as an antisulfotyrosine antibody mimic, demonstrating high affinity and specificity to sulfotyrosine through tailored selection schemes. Molecular docking simulations supported the characteristics of these SH2 mutants, enhancing the detection and enrichment of sulfoproteins.