Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands

Short contiguous peptides harboring proline-rich motifs are frequently invo lved in protein-protein interactions, such as associations with Src homolog y 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are dist inct, suggesting that additional protein families exist that use stacked ar omatic amino acids (AA domains) to bind polyproline motifs [1-3]. A polypro line motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytog enes, serves as a ligand for the Ena/VASP protein family - the vasodilator stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabl ed (Ena) and the Ena/VASP-like protein Evl [4-7], These share a similar ove rall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5], Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residue s within two conserved hydrophilic regions of the EVH1 domain abolished bin ding to ActA, Binding of an EVH1 mutant with reversed charges could partial ly be rescued by introducing complementary mutations within the ligand, Lik e SH3 domains, aromatic residues within the EVH1 domain interacted with pol yprolines, whereas the ligand specificity of either domain was determined b y reciprocally charged residues. The EVH1 domain is therefore a new additio n to the AA domain superfamily, which includes SH3 and WW domains.