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.

Keywords: E. coli; Ena/VASP; actin; biochemistry; chemical biology; epistasis; protein specificity; protein-protein interaction; short linear motif.

Figure 1.. Ena/VASP family EVH1 domains are highly conserved but differ in their affinity for a peptide from photoreceptor cilium actin regulator (PCARE).

(A) Sequence alignment of the EVH1 domains of ENAH, VASP, and EVL. Black denotes residues that are 100 % conserved; residues in gray share similar physicochemical properties. (B) Surface representation of ENAH EVH1 bound to an FP₄ peptide (PDB 1EVH) highlighting conservation among Ena/VASP paralogs. Residues shared between all three paralogs are white; residues shared by two paralogs are orange, residues that differ in each paralog are red. (C) Biolayer interferometry data and curves fit to determine the dissociation constants for peptide PCARE B binding to ENAH, EVL, or VASP EVH1 domains. Error reported as the standard deviation of two replicates.

Figure 2.. A photoreceptor cilium actin regulator (PCARE)-derived peptide selectively recruits ENAH in cells.

(A) Live MV^D7_shEVL cells expressing mRuby2-PCARE B with GFP, GFP-ENAH, GFP-dEVH1-ENAH, GFP-EVL, or GFP-VASP, imaged using total internal reflection fluorescence (TIRF) microscopy. Scale bar = 25 μm. (B) Enrichment ratio of mRuby2-PCARE B at GFP-positive focal adhesions over cytosolic signal under indicated overexpression conditions. n = 30 focal adhesions, N = 3 biological replicates. (C) Normalized fluorescence intensity of GFP signal (left axis) and mRuby2-PCARE B signal (right axis) along a line drawn through focal adhesions, indicated by the red arrow in (A). (D) Live MV^D7_shEVL cells expressing Mito-mRuby2-PCARE B with GFP, GFP-ENAH, GFP-dEVH1-ENAH, GFP-EVL, or GFP-VASP. Image is a maximum intensity projection of z-stack acquired using widefield fluorescence microscopy. Scale bar = 25 μm. (E) Enrichment ratio of GFP-tagged constructs to Mito-mRuby2-PCARE B-positive mitochondria over cytosolic signal under indicated overexpression conditions. n = 30 mitochondria, N = 3 biological replicates. (F) Magnified region of interest indicated by the box in (D), showing colocalization of Mito-mRuby2-PCARE B and GFP-ENAH at increasing depths in the cell. Scale bar = 5 μm. (G) Immunofluorescence labeling of MCF7 cells expressing nontargeting LKO control, ENAH-targeting shRNA, mRuby2-PCARE B, or Mito-mRuby2-PCARE B. Cells were fixed 8 hr after plating and immunolabeled for focal adhesion marker paxillin and endogenous ENAH, and additionally stained with phalloidin for F-actin. Box indicates positions of magnified regions of interest (ROI). Scale bar = 25 μm, magnified ROI scale bar = 5 μm. (H) Box-and-whisker plot of total paxillin-positive area per cell normalized to the total cell area, for indicated conditions. N = 3 biological replicates, n = 45–49 cells. (I) Violin plot of individual focal adhesion size (for adhesions greater than 0.25 μm²) for indicated conditions. The central black line indicates the median, peripheral gray lines indicate interquartile ranges. N = 3 biological replicates, n = 1452–2159 individual adhesions. In panels B, E, H, and I, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, Kruskal–Wallis test.

Figure 2—figure supplement 1.. Knockdown of EVL in MV^D7 cells.

Relative expression of MV^D7_shEVL vs. MV^D7_ntEVL (nontargeting control) is 42.8 ± 1.8 % by quantitative PCR (qPCR), three biological replicates.

Figure 2—figure supplement 2.. Expression of Ena/VASP proteins in MCF7 cells.

Representative western blot showing expression of ENAH, EVL, and VASP in MCF7 cells. At right, representative western blot showing the magnitude of knockdown using ENAH-targeted shRNA.

Figure 3.. ENAH EVH1 binds to a peptide from photoreceptor cilium actin regulator (PCARE) differently than to the peptide FPPPP.

(A) Surface representation of ENAH EVH1 bound to a segment of PCARE, highlighting Ena/VASP EVH1 domain conservation using colors as in Figure 1. (B) View comparing the orientations of an FP₄ peptide and the LP₄ region of PCARE. Side chains of the ENAH EVH1 domain are shown as sticks using tan for the FP₄ complex and gray for the PCARE complex. (C) Surface representation of ENAH EVH1 domain bound to the PCARE peptide. The LP₄ residues are light blue and other EVH1-interacting residues are green; insets show details of the interactions. Note that the PCARE^828–848 peptide is numbered as 133–153 in the PDB file.

Figure 4.. A peptide from photoreceptor cilium actin regulator (PCARE) achieves paralog selectivity by stabilizing an ENAH EVH1 domain-specific conformation.

(A) Superposition of ENAH EVH1 domains bound to PCARE or FP₄ peptide (PDB 1EVH). The black arrow highlights a 3 Å shift in a loop that forms part of the binding pocket. Insets show residues that differ between ENAH and VASP or EVL near this loop. (B) Lowest dTERMen energy obtained when swapping 0–6 residues from ENAH into EVL, when modeled on the structure of ENAH EVH1 bound to PCARE. * indicates the mutation was added based on manual inspection. (C) ENAH EVH1 domain bound to a peptide from PCARE, with residues that were swapped into the EVL EVH1 domain to rescue affinity marked as purple spheres. On the right are binding curves for WT EVL EVH1 domain and EVL_swapped EVH1 domain binding to PCARE B. Error reported as the standard deviation of two replicates.

Figure 4—figure supplement 1.. Fast protein liquid chromatography (FPLC) curves for EVL mutants.

EVL mutants run on a Superdex 75 column using fast protein liquid chromatography.

Figure 5.. Engineered peptides bind to the ENAH EVH1 domain with high affinity and specificity.

(A) Surface representation of ENAH EVH1 with binding sites discussed in this study indicated. (B) Design scheme for high-affinity binders ABI1-LPP and PCARE-Dual. (C) Biolayer interferometry (BLI)-binding and dissociation curves. Blue, orange, green, red, purple, and brown curves denote EVH1 concentrations in descending order. LPP: 80, 36, 16, 7.0, 3.1, and 1.4 μM. PCARE B and ABI1-LPP: 2.5, 1.3, 0.63, 0.31, 0.16, and 0.078 μM. PCARE-Dual: 0.50, 0.25, 0.0625, 0.031, 0.016, and 0.0078 μM. Values reported as k_diss ± SD for two independent BLI replicates. (D) BLI-binding curves for PCARE-Dual binding to the EVH1 domains of ENAH, VASP, or EVL. Errors for (B) and (D) are reported as the standard deviation of two replicates.

Figure 5—figure supplement 1.. Affinity of PCARE-Dual for ENAH vs. ENAH Y38E.

Affinities determined by biolayer interferometry (BLI) as described in the methods and in Hwang et al., 2021. Errors reported are the standard deviation of two to three replicates.