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 (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.
Cohen
Daniel M
DM
Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Chen
Hui
H
Johnson
Robert P
RP
Choudhury
Begum
B
Craig
Susan W
SW
D000199:Actins / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000737:chemistry; D022001:Focal Adhesions / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016608:Talin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016596:Vinculin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D001426:Bacterial Proteins; D005353:Fibronectins; D008164:Luminescent Proteins; D016608:Talin; C054562:yellow fluorescent protein, Bacteria; D016596:Vinculin; D000409:Alanine