A quantitative intracellular peptide binding assay reveals recognition determinants and context dependence of short linear motifs

Transient protein-protein interactions play key roles in controlling dynamic cellular responses. Many examples involve globular protein domains that bind to peptide sequences known as Short Linear Motifs (SLiMs), which are enriched in intrinsically disordered regions of proteins. Here we describe a novel functional assay for measuring SLiM binding, called Systematic Intracellular Motif Binding Analysis (SIMBA). In this method, binding of a foreign globular domain to its cognate SLiM peptide allows yeast cells to proliferate by blocking a growth arrest signal. A high-throughput application of the SIMBA method involving competitive growth and deep sequencing provides rapid quantification of the relative binding strength for thousands of SLiM sequence variants, and a comprehensive interrogation of SLiM sequence features that control their recognition and potency. We show that multiple distinct classes of SLiM-binding domains can be analyzed by this method, and that the relative binding strength of peptides in vivo correlates with their biochemical affinities measured in vitro. Deep mutational scanning provides high-resolution definitions of motif recognition determinants and reveals how sequence variations at non-core positions can modulate binding strength. Furthermore, mutational scanning of multiple parent peptides that bind human tankyrase ARC or YAP WW domains identifies distinct binding modes and uncovers context effects in which the preferred residues at one position depend on residues elsewhere. The findings establish SIMBA as a fast and incisive approach for interrogating SLiM recognition via massively parallel quantification of protein-peptide binding strength in vivo.