The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can’t be fully understood outside of their native context.

Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100 % conserved binding site. A proteome-wide screen revealed photoreceptor cilium actin regulator (PCARE) as a high-affinity ligand for ENAH EVH1. Here, we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind ENAH and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.

Protein interactions between intrinsically disordered, short linear motifs (SLiMs) and modular recognition domains are critical elements in many signal transduction pathways. Yet for most interactions, it is still unclear how low-complexity SLiMs discriminate between highly conserved but biologically distinct SLiM-binding proteins. The Ena/VASP family of proteins pose one such specificity problem. Paralogs ENAH, VASP, and EVL bind to a 5-residue SLiM prevalent in the proteome and perform distinct cellular functions despite sharing high sequence and structural similarity. To interrogate how the sequence context of SLiMs impacts Ena/VASP interactions, we performed an unbiased proteomic screen against the ENAH EVH1 domain. We discovered unexpected ways in which local and distal sequence elements flanking native SLiMs modulate binding. Particularly notable is a peptide from PCARE that achieves paralog specificity by stabilizing a unique conformation adopted only by ENAH. A PCARE-derived peptide can selectively recruit ENAH and block its activity in cells, establishing it as a valuable reagent to disentangle the roles of Ena/VASP paralogs and a potential agent for modulating ENAH function in breast cancer. Guided by our analyses of native interactions, we designed the tightest known ENAH EVH1 binder with a dissociation constant of 50 nM and 400-600-fold selectivity over EVL and VASP. Our work demonstrates that sequence context plays a prominent role in dictating SLiM interactions and can inform the design of custom molecules that target SLiM-based interactions.

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