Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin

Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (V-t) and residues 1 - 258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (V-h residues 710 - 836) and V-t. Charge-to-alanine mutations in V-t revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their K-d values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact V-t yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in K-d values between V-t binding to V-( 1 - 258), as opposed to full-length V-h-( 1 - 851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to V-h. Whereas V-t fails to compete with the talin rod domain for binding to V-( 1 - 258), competition occurs readily with full-length V-h, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-V-t interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-V-t interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-V-t and D4-V-t interfaces provide the high affinity ( similar to 10(-9)) autoinhibition observed in full-length vinculin.