Interaction of the N- and C-terminal domains of vinculin. Characterization and mapping studies

The vinculin head to tail intramolecular self-association controls its binding sites for other components of focal adhesions. To study this interaction, the head and tail domains were expressed, purified, and assayed for various characteristics of complex formation. Analytical centrifugation demonstrated a strong interaction in solution and formation of a complex more asymmetric than either of the individual domains. A survey of binding conditions using a solid-phase binding assay revealed characteristics of both electrostatic and hydrophobic forces involved in the binding. In addition, circular dichroism of the individual domains and the complex demonstrated that conformational changes likely occur in both domains during association. The interaction sites were more closely mapped on the protein sequence by deletion mutagenesis. Amino acids 181-226, a basic region within the acidic head domain, were identified as a binding site for the vinculin tail, and residues 1009-1066 were identified as sufficient for binding the head. Moreover, mutation of an acidic patch in the tail (residues 1013-1015) almost completely eliminated its ability to interact with the head domain further supporting the significance of ionic interactions in the binding. Our data indicate that the interaction between the head and tail domains of vinculin occurs through oppositely charged contact sites and results in conformational changes in both domains.