An integrative approach unveils a distal encounter site for rPTP8 and phospho-Src complex formation

The structure determination of protein tyrosine phosphatase (PTP): phospho-protein complexes, which is essential to understand how specificity is achieved at the amino acid level, remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPED1 and phospho-SrcKD as a model system, we have established an integrative workflow to address this problem, by means of which we generate a protein:phospho-protein complex model using predetermined protein struc-tures, SAXS and pTyr-tailored MD simulations. Our model reveals transient protein-protein interactions be-tween rPTPED1 and phospho-SrcKD and is supported by three independent experimental validations. Mea-surements of the association rate between rPTPED1 and phospho-SrcKD showed that mutations on the rPTPED1: SrcKD complex interface disrupts these transient interactions, resulting in a reduction in protein -pro-tein association rate and, eventually, phosphatase activity. This integrative approach is applicable to other PTP: phospho-protein complexes and the characterization of transient protein-protein interface interactions.