Unveiling an unexpected redox regulation of the folding, function and inhibition in the phosphotyrosine binding domain of FRS2.

Protein-protein interaction domains are essential for cellular homeostasis and the regulation of various molecular pathways, mediating highly specific and reversible binding events. The PhosphoTyrosine-Binding domains (PTB) play a pivotal role in regulating several cellular events, by recognizing phosphorylated and, in some cases, non-phosphorylated ligands. In this study we investigated the folding and functional properties of the PTB domain of FRS2 (Fibroblast growth factor receptor substrate 2) under oxidative and reductive experimental conditions. Results demonstrate a surprising and previously undetected role of a disulfide bond between Cys61 and Cys80 residues in such events. Through an extensive site-directed mutagenesis we demonstrated that the presence/absence of such disulfide bridge, although not changing dramatically the overall structure of the domain, significantly influence its dynamic properties by rewiring a subtle energetic network stabilizing the domain. These effects result in remodulating its binding properties with phosphorylated and unphosphorylated peptides. Molecular dynamics simulations further elucidated how the oxidative/reductive conditions modulate the dynamics of the domain. Finally, we identified lead inhibitory compounds with different efficacy observed across the oxidized and reduced states of the PTB domain. Altogether, these findings provide novel insights for understanding the mechanism of regulation of the function of the PTB domain of FRS2.