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

The vinculin head to tail intramolecular self-association controls its bind ing sites for other components of focal adhesions. To study this interactio n, the head and tail domains were expressed, purified, and assayed for vari ous characteristics of complex formation. Analytical centrifugation demonst rated a strong interaction in solution and formation of a complex more asym metric than either of the individual domains. A survey of binding condition s using a solid-phase binding assay revealed characteristics of both electr ostatic and hydrophobic forces involved in the binding. In addition, circul ar dichroism of the individual domains and the complex demonstrated that co nformational changes likely occur in both domains during association, The i nteraction sites were more closely mapped on the protein sequence by deleti on mutagenesis. Amino acids 181-226, a basic region within the acidic head domain, were identified as a binding site for the vinculin tail, and residu es 1009-1066 were identified as sufficient for binding the head. Moreover, mutation of an acidic patch in the tail (residues 1013-1015) almost complet ely eliminated its ability to interact with the head domain further support ing the significance of ionic interactions in the binding. Our data indicat e that the interaction between the head and tail domains of vinculin occurs through oppositely charged contact sites and results in conformational cha nges in both domains.