Short linear motifs, or SLiMs, mediate many protein interactions that assemble complexes and transduce signals in eukaryotes. Crystal structures of SLiMs bound to protein domains typically show just a few residues that are ordered at the interaction interface, raising the question of how specificity is achieved in such simple, low-information associations. We have been investigating the origins of SLiM binding specificity for the EVH1 domain, which is found in five families of human proteins. We are examining EVH1 domains from the Ena/VASP family of actin cytoskeleton regulating proteins and from HOMER1, a protein that provides scaffolding functions at the postsynaptic density. We have applied proteome peptide screening to identify binders of the EVH1 domains of these proteins. Analysis of Mena binders revealed three ways that sequence context enhances binding, including a novel conformational specificity mechanism that provides preferential peptide binding to one Ena/VASP family member over closely related paralogs. Mena and HOMER share a common ancestor, and we have examined how changes in the sequence and structure of the two related EVH1 domains have led to divergent yet still overlapping binding specificities. Our studies illustrate multiple mechanisms of contextual control in SLiM-mediated interactions and provide inspiration for engineering selective peptide binders of modular protein interaction domains that control important cellular processes.
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