Protein context shapes the specificity of domain-peptide interactionsin vivo

Abstract

SRC Homology 3 (SH3) domains contribute to cellular processes via their ability to support protein-protein interactions (PPIs). While the intrinsic binding specificities of SH3 domains have been studied in vitro , whether each domain is necessary and sufficient to define PPI specificity in vivo 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 that both the sequence of the SH3 host protein and the position of the SH3 domains within their host are critical for interaction specificity and for cellular processes such as endocytosis. We further validate these findings using a human multi-SH3 adaptor protein and investigating its ability to promote phase separation. Our work highlights the importance of the interplay between a SH3 domain and its host protein for the regulation of cellular and biophysical processes.