A distributed residue network permits conformational binding specificity in a conserved family of actin remodelers

• DOI: 10.7554/elife.70601
• Publication Date: 2021-12-02T16:02:32Z
• Authors: Theresa Hwang; Sara S Parker; Samantha M Hill; Meucci W Ilunga; Robert A Grant; Ghassan Mouneimne; Amy E Keating
• Keywords: epistasis; 0301 basic medicine; 572; QH301-705.5; Science; Molecular Conformation; protein-protein interaction; 03 medical and health sciences; Protein Domains; Biochemistry and Chemical Biology; Humans; Biology (General); Ena/VASP; Eye Proteins; 0303 health sciences; Binding Sites; protein specificity; Q; Microfilament Proteins; R; Phosphoproteins; short linear motif; Actins; HEK293 Cells; MCF-7 Cells; Medicine; actin; Cell Adhesion Molecules

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.