TY - JOUR
T1 - Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin
AU - Cohen, Daniel M.
AU - Chen, Hui
AU - Johnson, Robert P.
AU - Choudhury, Begum
AU - Craig, Susan W.
PY - 2005/4/29
Y1 - 2005/4/29
N2 - 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 (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 K d 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 V h. Whereas Vt 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-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 (∼10-9) autoinhibition observed in full-length vinculin.
AB - 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 (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 K d 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 V h. Whereas Vt 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-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 (∼10-9) autoinhibition observed in full-length vinculin.
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U2 - 10.1074/jbc.M414704200
DO - 10.1074/jbc.M414704200
M3 - Article
C2 - 15728584
AN - SCOPUS:20444492339
SN - 0021-9258
VL - 280
SP - 17109
EP - 17117
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -