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

Abstract

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.

Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH

“…Here we showed how defining the EVH1 binding motif as [FWYL]PXΦP is an over-simplification and how, by systematically examining the role of flanking sequences for just one EVH1 domain, we readily uncovered numerous examples in which the binding is modulated via additional extra-motif residues. Added to prior reports from investigations of individual interactions ( Stein and Aloy, 2008 ; Li et al, 2018a ; Aitio et al, 2010 ), our work definitively demonstrates the importance of sequence context on SLiM behavior by illustrating specific mechanisms, including an unusual conformational specificity mechanism that is documented in our companion paper ( Hwang et al, 2021 ). MassTitr provides a versatile experimental platform for uncovering context effects on domain-peptide interactions and will surely lead to similar insights into the recognition strategies of other domains.…”

“…Truncation experiments indicated that the 14-residues C-terminal of the LPPPP motif in PCARE are critical for its high affinity, hinting that extensive contacts between this region and the ENAH EVH1 domain could be responsible for the enhanced binding. Our subsequent work revealed the surprising structural basis for this affinity ( Hwang et al, 2021 ).…”

“…BLI was carried out as described in Hwang et al, 2021 . Briefly, biotinylated 6x-His-SUMO-peptide fusions were immobilized on streptavidin-coated tips and immersed into a concentration series of monomeric ENAH EVH1 domain diluted in BLI buffer (PBS pH 7.4, 1% BSA, 0.1% Tween-20, 1 mM DTT).…”

“…Monomeric EVH1 domain and small-scale preparations of biotinylated SUMO peptides used for BLI were purified as described in Hwang et al, 2021 .…”

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion

“…For example, EVH1-binding motifs might facilitate interaction with Protein-enabled homolog (ENAH) and Vasodilator stimulated phosphoprotein (VASP) PCARE interactor proteins that we have identified in yeast two-hybrid screening and tandem affinity purification studies ( 9 ). A very recent structural study has shown that PCARE binds with a high affinity to the EVH1 domain of the protein ENAH, which as noted we had previously identified as a PCARE interactor, that plays a role in actin polymerization and cytoskeletal remodelling ( 29 ). Taken together, these studies establish that PCARE acts through ENAH to regulate actin filament assembly in the eye photoreceptor cilium.…”