Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

Abstract

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Fig. 1. Superposition of the backbone (N, C_α and C′) atoms for the 15 lowest energy structures of the VASP EVH1 domain. Core residues are shown in orange and the residues of the aromatic triad, Tyr16, Trp23, Phe79 are shown in red. Figures were created using the program Molscript (Kraulis, 1991). The structural statistics are given in Table I.

Fig. 2. Mutational analysis of the native ActA-derived ₃₃₂SFEFPPPPTEDEL₃₄₄ peptide by screening of the VASP EVH1 domain against cellulose membrane-bound ActA peptide derivatives. Residues in the peptides were substituted each in turn by all 20 l-amino acids (rows) and assayed for binding to the EVH1 domain of human VASP. The left column is a control, showing the binding of the wild-type sequence. Each of the other positions in the grid represent peptides synthesized with single amino acid substitutions, with the spot intensities reflecting the binding affinities of each substituted peptide. Quantitative measurements of binding constants were later determined using fluorescence spectroscopy based on the results of these analyses.

Fig. 3. Bar plot showing changes in chemical shifts of the backbone ¹H and ¹⁵N resonances between the unbound VASP EVH1 domain and the domain following titration to an equimolar ratio with each of the three ActA-derived peptides discussed in the text. Shifts are displayed as a function of sequence position. Yellow bars represent the changes seen with addition of ₃₃₂SFEFPPPPTEDEL₃₄₄, pink bars with the shorter ₃₃₄EFPPPPTEDEL₃₄₄, and black bars with the C-terminally truncated peptide ₃₃₄EFPPPPTED₃₄₂. For clarity, the regions of interest (Gln31, Ala32 and Ala85, Arg86) are enlarged and displayed below the full sequence plot. The perturbations, measured from ¹⁵N-HSQC spectra, are shown as a combined, weighted function of ¹⁵N and ¹H shifts: Δδ_TOTAL = Δδ[¹H] + 0.2*Δδ[¹⁵N] (Hadjuk et al., 1997). Changes in chemical shifts of the sidechain protons of the peptide, measured from ¹³C-(ω1)–¹³C-(ω2) double half-filtered ¹H TOCSY spectra (Gemmecker et al., 1992; Ikura and Bax, 1992) on a sample containing fully ¹³C,¹⁵N-labelled protein bound to unlabelled peptide in a D₂O solvent, are shown in Table III.

Fig. 4. (A) Combined chemical shift perturbations of residues in the EVH1 domain on binding to ActA SFEFPPPPTEDEL. Red: perturbations >0.4 p.p.m.; purple: perturbations between 0.25 and 0.4 p.p.m.; lilac: perturbations between 0.1 and 0.25 p.p.m. Spheres denote ¹⁵N/¹H pairs which experience the greatest differences in chemical shift perturbation when either the peptide ₃₄₃EL₃₄₄ or ₃₃₂SF₃₃₃ residues are removed (specifically, these are the backbone N/H atoms of the EVH1 Gln31, Ala32 and Ala85, Arg86, respectively). The reduction in shift perturbations of the Trp23 indole N/H atoms result from the much weaker overall binding to a peptide lacking the ‘EL’ epitope. (B) Chemical shift perturbations of the ActA SFEFPPPPTEDEL peptide upon EVH1 binding (see Table III). The surface of the VASP EVH1 domain is coloured according to (A), with the difference that only surface exposed amides are shown. Residues in the peptide are coloured cyan (perturbations >0.2 p.p.m.) and green (perturbations between 0.15 and 0.2 p.p.m.). (C) and (D) show the hydrophobic surfaces of VASP and Mena EVH1 domains, respectively. It should be noted that the Mena EVH1 domain was co-crystallized using a much shorter peptide comprising just the core FPPPPT residues (Prehoda et al., 1999). The view of each molecule is rotated 45° forwards about the x-axis, with respect to the view in (A) in order to show the additional binding contacts of the longer peptide. Hydrophobic surfaces were calculated with the program GRASP (Nicholls et al., 1991) using hydrophobicity scales from Covell et al. (1994). Hydrophobic areas are shown in yellow and hydrophilic areas in purple. The yellow hydrophobic groove, running from top to bottom of the molecule, comprises the binding site for the ActA peptide. Contacts between the FPPPP motif and the residues in and around the triad region, Tyr16, Trp23, Phe79 and Met14 are clearly visible, as are secondary contacts between the peptide C-terminal leucine and Met54, Ala32 and Phe33 of the protein. The residues comprising the groove in each of the proteins are labelled, showing the distribution of surface hydrophobicities to be highly similar between the VASP and Mena domains. The SFEFPPPPTEDEL peptide was docked onto the domain as described in the Materials and methods.

Fig. 4. (A) Combined chemical shift perturbations of residues in the EVH1 domain on binding to ActA SFEFPPPPTEDEL. Red: perturbations >0.4 p.p.m.; purple: perturbations between 0.25 and 0.4 p.p.m.; lilac: perturbations between 0.1 and 0.25 p.p.m. Spheres denote ¹⁵N/¹H pairs which experience the greatest differences in chemical shift perturbation when either the peptide ₃₄₃EL₃₄₄ or ₃₃₂SF₃₃₃ residues are removed (specifically, these are the backbone N/H atoms of the EVH1 Gln31, Ala32 and Ala85, Arg86, respectively). The reduction in shift perturbations of the Trp23 indole N/H atoms result from the much weaker overall binding to a peptide lacking the ‘EL’ epitope. (B) Chemical shift perturbations of the ActA SFEFPPPPTEDEL peptide upon EVH1 binding (see Table III). The surface of the VASP EVH1 domain is coloured according to (A), with the difference that only surface exposed amides are shown. Residues in the peptide are coloured cyan (perturbations >0.2 p.p.m.) and green (perturbations between 0.15 and 0.2 p.p.m.). (C) and (D) show the hydrophobic surfaces of VASP and Mena EVH1 domains, respectively. It should be noted that the Mena EVH1 domain was co-crystallized using a much shorter peptide comprising just the core FPPPPT residues (Prehoda et al., 1999). The view of each molecule is rotated 45° forwards about the x-axis, with respect to the view in (A) in order to show the additional binding contacts of the longer peptide. Hydrophobic surfaces were calculated with the program GRASP (Nicholls et al., 1991) using hydrophobicity scales from Covell et al. (1994). Hydrophobic areas are shown in yellow and hydrophilic areas in purple. The yellow hydrophobic groove, running from top to bottom of the molecule, comprises the binding site for the ActA peptide. Contacts between the FPPPP motif and the residues in and around the triad region, Tyr16, Trp23, Phe79 and Met14 are clearly visible, as are secondary contacts between the peptide C-terminal leucine and Met54, Ala32 and Phe33 of the protein. The residues comprising the groove in each of the proteins are labelled, showing the distribution of surface hydrophobicities to be highly similar between the VASP and Mena domains. The SFEFPPPPTEDEL peptide was docked onto the domain as described in the Materials and methods.

Fig. 4. (A) Combined chemical shift perturbations of residues in the EVH1 domain on binding to ActA SFEFPPPPTEDEL. Red: perturbations >0.4 p.p.m.; purple: perturbations between 0.25 and 0.4 p.p.m.; lilac: perturbations between 0.1 and 0.25 p.p.m. Spheres denote ¹⁵N/¹H pairs which experience the greatest differences in chemical shift perturbation when either the peptide ₃₄₃EL₃₄₄ or ₃₃₂SF₃₃₃ residues are removed (specifically, these are the backbone N/H atoms of the EVH1 Gln31, Ala32 and Ala85, Arg86, respectively). The reduction in shift perturbations of the Trp23 indole N/H atoms result from the much weaker overall binding to a peptide lacking the ‘EL’ epitope. (B) Chemical shift perturbations of the ActA SFEFPPPPTEDEL peptide upon EVH1 binding (see Table III). The surface of the VASP EVH1 domain is coloured according to (A), with the difference that only surface exposed amides are shown. Residues in the peptide are coloured cyan (perturbations >0.2 p.p.m.) and green (perturbations between 0.15 and 0.2 p.p.m.). (C) and (D) show the hydrophobic surfaces of VASP and Mena EVH1 domains, respectively. It should be noted that the Mena EVH1 domain was co-crystallized using a much shorter peptide comprising just the core FPPPPT residues (Prehoda et al., 1999). The view of each molecule is rotated 45° forwards about the x-axis, with respect to the view in (A) in order to show the additional binding contacts of the longer peptide. Hydrophobic surfaces were calculated with the program GRASP (Nicholls et al., 1991) using hydrophobicity scales from Covell et al. (1994). Hydrophobic areas are shown in yellow and hydrophilic areas in purple. The yellow hydrophobic groove, running from top to bottom of the molecule, comprises the binding site for the ActA peptide. Contacts between the FPPPP motif and the residues in and around the triad region, Tyr16, Trp23, Phe79 and Met14 are clearly visible, as are secondary contacts between the peptide C-terminal leucine and Met54, Ala32 and Phe33 of the protein. The residues comprising the groove in each of the proteins are labelled, showing the distribution of surface hydrophobicities to be highly similar between the VASP and Mena domains. The SFEFPPPPTEDEL peptide was docked onto the domain as described in the Materials and methods.

Fig. 4. (A) Combined chemical shift perturbations of residues in the EVH1 domain on binding to ActA SFEFPPPPTEDEL. Red: perturbations >0.4 p.p.m.; purple: perturbations between 0.25 and 0.4 p.p.m.; lilac: perturbations between 0.1 and 0.25 p.p.m. Spheres denote ¹⁵N/¹H pairs which experience the greatest differences in chemical shift perturbation when either the peptide ₃₄₃EL₃₄₄ or ₃₃₂SF₃₃₃ residues are removed (specifically, these are the backbone N/H atoms of the EVH1 Gln31, Ala32 and Ala85, Arg86, respectively). The reduction in shift perturbations of the Trp23 indole N/H atoms result from the much weaker overall binding to a peptide lacking the ‘EL’ epitope. (B) Chemical shift perturbations of the ActA SFEFPPPPTEDEL peptide upon EVH1 binding (see Table III). The surface of the VASP EVH1 domain is coloured according to (A), with the difference that only surface exposed amides are shown. Residues in the peptide are coloured cyan (perturbations >0.2 p.p.m.) and green (perturbations between 0.15 and 0.2 p.p.m.). (C) and (D) show the hydrophobic surfaces of VASP and Mena EVH1 domains, respectively. It should be noted that the Mena EVH1 domain was co-crystallized using a much shorter peptide comprising just the core FPPPPT residues (Prehoda et al., 1999). The view of each molecule is rotated 45° forwards about the x-axis, with respect to the view in (A) in order to show the additional binding contacts of the longer peptide. Hydrophobic surfaces were calculated with the program GRASP (Nicholls et al., 1991) using hydrophobicity scales from Covell et al. (1994). Hydrophobic areas are shown in yellow and hydrophilic areas in purple. The yellow hydrophobic groove, running from top to bottom of the molecule, comprises the binding site for the ActA peptide. Contacts between the FPPPP motif and the residues in and around the triad region, Tyr16, Trp23, Phe79 and Met14 are clearly visible, as are secondary contacts between the peptide C-terminal leucine and Met54, Ala32 and Phe33 of the protein. The residues comprising the groove in each of the proteins are labelled, showing the distribution of surface hydrophobicities to be highly similar between the VASP and Mena domains. The SFEFPPPPTEDEL peptide was docked onto the domain as described in the Materials and methods.