How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

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Abstract
Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. For cell survival, most eukaryotic cells need to be mechanically anchored to their environment. This is done by transmembrane proteins, including integrins, which externally bind to the extracellular matrix and on the cell interior to the contractile cytoskeleton via scaffolding proteins. One essential scaffolding protein is talin, which binds to integrins via its head and to the cytoskeletal filament f-actin via its rodlike tail. As cells apply tensile forces to newly formed adhesion sites, proteins that are part of such force-bearing networks get stretched and might change their structure and thus function. One of many proteins that are recruited to newly formed adhesions is vinculin, and vinculin recruitment is upregulated by tensile mechanical force—but how? Since talin's vinculin binding sites are buried in its native structure, we used steered molecular dynamics here to derive a high resolution structural model of how tensile mechanical forces might activate talin's vinculin binding sites. Once tensile forces break up the talin rod into helix subbundles, an event that we find here to constitute the main energy barrier, we propose how the strain-induced gradual exposure of the vinculin-binding helices finally allows for their activation and enables helix swapping with the vinculin head.