Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation

The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1–R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1–R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ∼5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression.