The specificity of SRC Homology 3 (SH3) domain interactions is modulated by their protein context in vivo

Many cell signaling pathways and cellular processes depend on the binding specificity of SRC Homology 3 (SH3) domains. While the intrinsic binding specificities of SH3 domains have been studied intensively , whether each domain is necessary and sufficient to define protein-protein interaction (PPI) specificity is largely unknown. We combine genome editing, deep mutagenesis scanning, PPI mapping, growth phenotyping and single cell imaging in yeast to identify SH3-dependent PPI networks and their associated phenotypes. We show by domain deletion, swapping and shuffling that the sequence of the SH3’s host protein and the position of the SH3 domains are critical for interaction specificity, and for the proper function of cellular processes such as endocytosis. We further show that these findings apply to a human SH3 adaptor protein and to its ability to promote phase separation. Our work highlights the importance of the interplay between a SH3 domain and its host protein as well as its positioning for the regulation of cellular and biophysical processes.