Residues neighboring an SH3-binding motif participate in the interaction in vivo.

In signaling networks, protein-protein interactions are often mediated by modular domains that bind short linear motifs. The motifs' sequences affect many factors, among them affinity and specificity, or the ability to bind strongly and to the appropriate partners. Using Deep Mutational Scanning to create a mutant library, and protein complementation assays to measure protein-protein interactions, we determined the in vivo binding strength of a library of mutants of a binding motif on the MAP kinase kinase Pbs2, which binds the SH3 domain of the osmosensor protein Sho1 in Saccharomyces cerevisiae. These measurements were made using the full-length endogenous proteins in their native cellular environment. We find that, along with residues within the canonical motif, many mutations in the residues neighboring the motif also modulate binding strength. Interestingly, all Pbs2 mutations that increase binding are situated outside of the Pbs2 region that interacts with the canonical SH3-binding pocket, suggesting that other surfaces on Sho1 contribute to binding. We use predicted structures and mutations to propose a model of binding that involves residues neighboring the canonical Pbs2 motif binding outside of the canonical SH3 binding pocket. We compared this predicted structure with known structures of SH3 domains binding peptides through residues outside of the motif, and put forth possible mechanisms through which Pbs2 can bind specifically to Sho1. We propose that for certain SH3 domain-motif pairs, affinity and specificity are determined by a broader range of sequences than what has previously been considered, potentially allowing easier differentiation between otherwise similar partners.