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 speci®cally 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 af®nities of these EVH1±peptide interactions are strongly dependent on the recognition of residues¯anking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K d s bȳ uorescence titration, and NMR chemical shift mapping, revealed a second af®nity-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 af®nity 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.

Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the 'ring spot' effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm2 as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.

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