Structural basis for amplifying vinculin activation by talin

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh.VBS1 complex, a validated model of the Vh.VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.     

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

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.     

α-Catenin uses a novel mechanism to activate vinculin

Vinculin, an actin-binding protein, is emerging as an important regulator of adherens junctions. In focal-adhesions, vinculin is activated by simultaneous binding of talin to its head domain and actin filaments to its tail domain. Talin is not present in adherens junctions. Consequently, the identity of the ligand that activates vinculin in cell-cell junctions is not known. Here we show that in the presence of F-actin, α-catenin, a cytoplasmic component of the cadherin adhesion complex, activates vinculin. Direct binding of α-catenin to vinculin is critical for this event because a point mutant (α-catenin L344P) lacking high affinity binding does not activate vinculin. Furthermore, unlike all known vinculin activators, α-catenin binds to and activates vinculin independently of an A50I substitution in the vinculin head, a mutation that inhibits vinculin binding to talin and IpaA. Collectively, these data suggest that α-catenin employs a novel mechanism to activate vinculin and may explain how vinculin is differentially recruited and/or activated in cell-cell and cell-matrix adhesions.     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the alpha-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS alpha-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.     

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

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.     

A vinculin binding domain from the talin rod unfolds to form a complex with the vinculin head

The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds beta integrins, is linked to an extended rod having a C-terminal actin binding site and several vinculin binding sites (VBSs). The NMR structure of residues 755-889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to vinculin. Interestingly, the talin 755-889 fragment binds more than one vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.     

Subcellular localization of talin is regulated by inter-domain interactions

Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482-787)), and the THD restrains talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the talin rod domain that regulate the subcellular localization of talin.     

Activation of vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.     

Vinculin binding in its closed conformation by a helix addition mechanism

Vinculin links integrin receptors to the actin cytoskeleton by binding to talin. Vinculin is held in an inactive, closed-clamp conformation through hydrophobic interactions between its head and tail domains, and vinculin activation has long been thought to be dependent upon severing the head-tail interaction. Talin, alpha-actinin, and the invasin IpaA of Shigella flexneri sever vinculin's head-tail interaction by inserting an alpha-helix into vinculin's N-terminal four-helical bundle, provoking extensive conformational changes by a helical bundle conversion mechanism; these alterations in vinculin structure displace its tail domain, allowing vinculin to bind to its other partners. IpaA harbors two juxtaposed alpha-helical vinculin-binding sites (VBS) in its C-terminus. Here, we report that the lower affinity VBS of IpaA can also bind to the adjacent C-terminal four-helical bundle of vinculin's head domain through a helix addition mechanism. These hydrophobic interactions do not alter the conformation of this helical bundle, and the architecture of the complex suggests that IpaA can simultaneously interact with both of the four-helical bundle domains of vinculin's N-terminus to stabilize vinculin-IpaA interactions.     

Three-dimensional structure of vinculin bound to actin filaments

Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. We used a combination of electron microscopy, computational docking, and biochemistry to provide an atomic model of how the vinculin tail binds actin filaments. The vinculin tail actin binding site comprises two distinct regions. One of these regions is exposed in the full-length autoinhibited conformation of vinculin, whereas the second site is sterically occluded by vinculin's N-terminal domain. The partial accessibility of the F-actin binding site in the autoinhibited full-length vinculin structure suggests that F-actin can act as part of a combinatorial input framework with other binding partners such as alpha-catenin or talin to induce vinculin head-tail dissociation, thus promoting vinculin activation. Furthermore, binding to F-actin potentiates a local rearrangement in the vinculin tail that in turn promotes vinculin dimerization and, hence, formation of actin bundles.     

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.     

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

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.     

Vinculin activation is necessary for complete talin binding

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.     

RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.     

Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin

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 (Vt) 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 Kd 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 Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length Vh, 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 (approximately 10(-9)) autoinhibition observed in full-length vinculin.     

A Molecular Dynamics Investigation of Vinculin Activation

Vinculin activation plays a critical role in focal adhesion initiation and formation. In its native state, vinculin is in an autoinhibitory conformation in which domain 1 prevents interaction of the vinculin tail domain with actin by steric hindrance. Once activated, vinculin is able to interact with both actin and talin. Several hypotheses have been put forth addressing the mechanisms of vinculin activation. One set of studies suggests that vinculin interaction with talin is sufficient to cause activation, whereas another set of studies suggests that a simultaneous interaction with several binding partners is necessary to achieve vinculin activation. Using molecular-dynamics (MD) simulations, we investigate the mechanisms of vinculin activation and suggest both a trajectory of conformational changes leading to vinculin activation, and key structural features that are likely involved in stabilizing the autoinhibited conformation. Assuming that the simultaneous interaction of vinculin with both actin and talin causes a stretching force on vinculin, and that vinculin activation results from a removal of steric hindrance blocking the actin-binding sites, we simulate with MD the stretching and activation of vinculin. The MD simulations are further confirmed by normal-mode analysis and simulation after residue modification. Taken together, the results of these simulations suggest that bending of the vinculin-binding-site region in vinculin away from the vinculin tail is the likely trajectory of vinculin activation.     

Structural and dynamic characterization of a vinculin binding site in the talin rod

Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction.     

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr¹⁰⁶⁵ on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr¹⁰⁶⁵ in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr¹⁰⁶⁵ (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr¹⁰⁶⁵ phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr¹⁰⁶⁵ phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.     

The Interaction of Vinculin with Actin

Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion.