The Vinculin D1 Domain Stabilizes αE-Catenin in a Strong Actin Binding State

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Cadherins are an ancient class of transmembrane proteins that mediate intercellular adhesion. Previous work from our laboratory and others established that the E-cadherin cytoplasmic tail forms a ternary complex with αE-catenin and β-catenin, and that αE-catenin forms a force-sensitive catch bond to filamentous (F)-actin as part of this complex. Biochemical and cell biological data indicate that αE-catenin can also bind and recruit the actin-binding protein vinculin to intercellular junctions, where vinculin is proposed to reinforce the connection between E-cadherin and the actin cytoskeleton. In this study we investigated how conformational changes in αE-catenin are coupled to vinculin binding, and how vinculin binding alters the force-dependent interaction of αE-catenin with actin. Using a single-molecule optical trapping assay, we found that the addition of the vinculin D1 domain, which cannot bind actin, to the ternary E-cadherin/αE-catenin/β-catenin complex traps the resulting quaternary complex in a constitutively strong-binding state with dramatically longer binding lifetimes to actin at low force relative to the ternary cadherin-catenin complex. In addition, we used isothermal calorimetry and point mutagenesis to probe the structural transitions that mediate binding of vinculin to αE-catenin. We found that the R551A point mutation, which eliminates a salt bridge between the MI and MIII domains of αE-catenin, resulted in a 100-fold increase in the affinity of αE-catenin for the vinculin D1 domain relative to that of wild-type αE-catenin. These and other data suggest that mutual activation of vinculin and αE-catenin traps both molecules in conformational states with enhanced affinities for actin, providing a potential mechanism by which relatively small changes in force experienced by the cadherin-catenin complex can be amplified into large changes in adhesion stability.