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

J Biol Chem. 2005 Apr 29;280(17):17109-17. doi: 10.1074/jbc.M414704200. Epub 2005 Feb 22.


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 (Vt) and residues 1-258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (Vh residues 710-836) and Vt. Charge-to-alanine mutations in Vt revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their Kd values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact Vt yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in Kd values between Vt binding to V-(1-258), as opposed to full-length Vh-(1-851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length Vh, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-Vt 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-Vt interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-Vt and D4-Vt interfaces provide the high affinity (approximately 10(-9)) autoinhibition observed in full-length vinculin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / chemistry
  • Alanine / chemistry
  • Amino Acid Sequence
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Binding, Competitive
  • Cell Line
  • Dose-Response Relationship, Drug
  • Fibronectins / chemistry
  • Focal Adhesions / metabolism
  • Genetic Vectors
  • Humans
  • Immunoblotting
  • Kinetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Talin / chemistry
  • Talin / genetics
  • Talin / metabolism*
  • Transfection
  • Vinculin / chemistry
  • Vinculin / genetics
  • Vinculin / metabolism*


  • Actins
  • Bacterial Proteins
  • Fibronectins
  • Luminescent Proteins
  • Talin
  • yellow fluorescent protein, Bacteria
  • Vinculin
  • Alanine