A CONSERVED MOTIF IN THE TAIL DOMAIN OF VINCULIN MEDIATES ASSOCIATIONWITH AND INSERTION INTO ACIDIC PHOSPHOLIPID-BILAYERS

The tail domain of vinculin (V-t) contains a salt-insensitive binding site for acidic phospholipids which is masked by the intramolecular he ad-tail interaction in native vinculin [Johnson, R. P., and Craig, S. W. (1995) Biochem. Biophys. Res. Commun. 210, 159-164]. To characteriz e further this phospholipid binding site, we have used hydrophobic pho tolabeling with a photoactivatable phosphatidylcholine analogue to det ect insertion of protein into the lipid bilayer. We show here that, al though the properties of binding to acidic phospholipid vesicles and s pontaneous insertion into the bilayer are cryptic and inactive in vinc ulin at physiologic ionic strength, these activities of the purified t ail domain can be activated by physical and chemical disruption of the intramolecular interaction between the head and tail domains. By anal yzing the lipid binding and insertion activity of a series of GST-V-t fusion proteins, we defined 55 amino acids, comprising vinculin residu es 916-970, that mimic the lipid-binding and insertion activity of V-t . Predictions of secondary structure suggest that these 55 amino acids form a basic, amphipathic helical hairpin. This prediction is support ed by circular dichroism analysis, which indicates that at least 80% o f the residues in residues 916-970 are in a helical conformation. This predicted helical hairpin motif, which is conserved in all vinculins and is present in an acidic phospholipid-binding region of alpha-caten in, is distinct from C2 and PH domains, and likely represents a third type of acidic phospholipid-binding structure.