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 ). 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 (). 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 () using hydrophobicity scales from . 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.