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

ABSTRACT: 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.

SUBMITTER: Yao M

PROVIDER: S-EPMC3980218 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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

Yao Mingxi M Goult Benjamin T BT Chen Hu H Cong Peiwen P Sheetz Michael P MP Yan Jie J

Scientific reports 20140409

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 refoldin ...[more]

PMID: 24714394

Similar Datasets

Vinculin activation is necessary for complete talin binding.

Project description:Focal adhesions are critical to a number of cellular processes that involve mechanotransduction and mechanical interaction with the cellular environment. The growth and strengthening of these focal adhesions is dependent on the interaction between talin and vinculin. This study investigates said interaction and how vinculin activation influences it. Using molecular dynamics, the interaction between talin's vinculin binding site (VBS) and vinculin's domain 1 (D1) is simulated both before and after vinculin activation. The simulations of VBS binding to vinculin before activation suggest the proximity of the vinculin tail to D1 prevents helical movement in D1 and thus prevents binding of VBS. In contrast, interaction of VBS with activated vinculin shows the possibility of complete VBS insertion into D1. In the simulations of both activated and autoinhibited vinculin where VBS fails to fully bind, VBS demonstrates significant hydrophobic interaction with surface residues in D1. These interactions link VBS to D1 even without its proper insertion into the hydrophobic core. Together these simulations suggest VBS binds to vinculin with the following mechanism: 1), VBS links to D1 via surface hydrophobic interactions; 2), vinculin undergoes activation and D1 is moved away from the vinculin tail; 3), helices in D1 undergo conformational change to allow VBS binding; and 4), VBS inserts itself into the hydrophobic core of D1.

| S-EPMC3021670 | biostudies-literature

Allosteric activation of vinculin by talin.

Project description:The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

| S-EPMC10354202 | biostudies-literature

Molecular dynamics study of talin-vinculin binding.

Project description:Cells can sense mechanical force in regulating focal adhesion assembly. One vivid example is the force-induced recruitment of vinculin to reinforce initial contacts between a cell and the extracellular matrix. Crystal structures of the unbound proteins and bound complex between the vinculin head subdomain (Vh1) and the talin vinculin binding site 1 (VBS1) indicate that vinculin undergoes a conformational change upon binding to talin. However, the molecular basis for this event and the precise nature of the binding pathway remain elusive. In this article, molecular dynamics is used to investigate the binding mechanism of Vh1 and VBS1 under minimal constraints to facilitate binding. One simulation demonstrates binding of the two molecules in the complete absence of external force. VBS1 makes early hydrophobic contact with Vh1 by positioning the critical hydrophobic residues (L608, L615, and L622) in the groove formed by helices 1 and 2 of Vh1. The solvent-exposed hydrophobic residues (V619 and L623) then gradually penetrate the hydrophobic core of Vh1, thus further separating helix 1 from helix 2. These critical residues are highly conserved as large hydrophobic side groups in other vinculin binding sites; studies also have demonstrated that these residues are essential in Vh1-VBS1 binding. Similar binding mechanisms are also demonstrated in separate molecular dynamics simulations of Vh1 binding to other vinculin binding sites both in talin and alpha-actinin.

| S-EPMC2483755 | biostudies-literature

Stretching single talin rod molecules activates vinculin binding.

Project description:The molecular mechanism by which a mechanical stimulus is translated into a chemical response in biological systems is still unclear. We show that mechanical stretching of single cytoplasmic proteins can activate binding of other molecules. We used magnetic tweezers, total internal reflection fluorescence, and atomic force microscopy to investigate the effect of force on the interaction between talin, a protein that links liganded membrane integrins to the cytoskeleton, and vinculin, a focal adhesion protein that is activated by talin binding, leading to reorganization of the cytoskeleton. Application of physiologically relevant forces caused stretching of single talin rods that exposed cryptic binding sites for vinculin. Thus in the talin-vinculin system, molecular mechanotransduction can occur by protein binding after exposure of buried binding sites in the talin-vinculin system. Such protein stretching may be a more general mechanism for force transduction.

| S-EPMC9339221 | biostudies-literature

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.

Project description:The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482-789. Talin 482-655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656-789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482-655 fold. We demonstrate that the talin 482-655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

| S-EPMC514914 | biostudies-literature

Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin.

Project description:The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β-integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F-actin through the agency of its five-helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α-helices coined vinculin binding sites (VBSs) that reside within numerous four- and five-helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N-terminal seven-helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head-tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five-helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2-vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.

| S-EPMC3375758 | biostudies-literature

Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy.

Project description:The dimeric focal adhesion protein talin contains up to 22 cryptic vinculin binding sites that are exposed by unfolding. Using a novel method to monitor the in situ dynamics of the talin dimer stretch, we find that in contrast to several prevalent talin dimer models the integrin-binding talin N-termini are separated by 162 ± 44 nm on average whereas as expected the C-terminal dimerization domains colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655 label, we found that optimal vinculin and vinculin head binding occurred when talin was stretched to 180 nm, while the controls did not bind to talin. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. We suggest that talin stretches as an antiparallel dimer and that activates vinculin binding in a cooperative manner, consistent with the stabilization of folded talin by other binding proteins.

| S-EPMC5367886 | biostudies-literature

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin.

Project description:The cytoskeletal proteins talin and vinculin are localized at cell-matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F-actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four- and five-helix bundles that harbor amphipathic α-helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four-helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α-helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four-helix bundle.

| S-EPMC3189532 | biostudies-literature

Mechanical stability of αT-catenin and its activation by force for vinculin binding.

Project description:αT (Testes)-catenin, a critical factor regulating cell-cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell-cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates αT-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of αT-catenin and determined how force impacts vinculin binding to αT-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at ∼6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that αT-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of αT-catenin and how αT-catenin regulates cell-cell adhesion at the cardiomyocyte ICD.

| S-EPMC6727763 | biostudies-literature

Direct single-molecule quantification reveals unexpectedly high mechanical stability of vinculin-talin/α-catenin linkages.

Project description:The vinculin-mediated mechanosensing requires establishment of stable mechanical linkages between vinculin to integrin at focal adhesions and to cadherins at adherens junctions through associations with the respective adaptor proteins talin and α-catenin. However, the mechanical stability of these critical vinculin linkages has yet to be determined. Here, we developed a single-molecule detector assay to provide direct quantification of the mechanical lifetime of vinculin association with the vinculin binding sites in both talin and α-catenin, which reveals a surprisingly high mechanical stability of the vinculin-talin and vinculin-α-catenin interfaces that have a lifetime of >1000 s at forces up to 10 pN and can last for seconds to tens of seconds at 15 to 25 pN. Our results suggest that these force-bearing intermolecular interfaces provide sufficient mechanical stability to support the vinculin-mediated mechanotransduction at cell-matrix and cell-cell adhesions.

| S-EPMC6920023 | biostudies-literature

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