Pathways and Intermediates in Forced Unfolding of Spectrin Repeats

Altmann, Stephan M.; Grünberg, Raik; Lenne, Pierre-François; Ylänne, Jari; Raae, Arnt J.; Herbert, Kristina M.; Saraste, Matti; Nilges, Michaël; Hörber, J. K. H.

doi:10.1016/s0969-2126(02)00808-0

Cited by 74 publications

(82 citation statements)

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“…In ␣-actinin, these repeats are aligned in a symmetric fashion that allows for the formation of the rigid dimer through interactions of the R1 and R4 repeats and of the R2 and R3 repeats (12,40). However, spectrin repeats can also form stable unfolded intermediates when subjected to mechanical stress (1), as occurs following the formation of adhesion complexes, and the spectrin repeats of ␣-actinin also harbor docking sites for a number of other cytoskeletal proteins (32), in particular vinculin (31).…”

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confidence: 99%

Structural Dynamics of α-Actinin-Vinculin Interactions

Bois

Borgon

Vonrhein

et al. 2005

Molecular and Cellular Biology

123

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␣-Actinin is a ubiquitously expressed and essential cytoskeletal protein that cross-links actin filaments at sites of cell-cell (adherens junctions) and cell-matrix (focal adhesion) junctions and at the leading edges of the cell membranes of migrating cells. ␣-Actinin is a member of a family of structurally related proteins, including spectrin, dystrophin, and utrophin, which regulate the organization of the actin cytoskeleton in a celltype-specific fashion (32). These proteins have a globular structure, with calponin homology (CH) domains at their N termini, a central rod domain containing ␣-helical spectrin repeats, and a C-terminal domain that contains calmodulin-like domains harboring EF-hand motifs (7).␣-Actinin is a rod-shaped antiparallel dimer of two 100-kDa monomers; this configuration positions its actin-binding CH motifs at either end of the rigid rod, an arrangement that allows ␣-actinin to efficiently cross-link actin filaments into tight bundles (12,40). The central rod domain of ␣-actinin contains four spectrin repeats (R1 to R4), which are triplehelical coiled-coil bundles that are connected by helical linkers (7,12). In ␣-actinin, these repeats are aligned in a symmetric fashion that allows for the formation of the rigid dimer through interactions of the R1 and R4 repeats and of the R2 and R3 repeats (12, 40). However, spectrin repeats can also form stable unfolded intermediates when subjected to mechanical stress (1), as occurs following the formation of adhesion complexes, and the spectrin repeats of ␣-actinin also harbor docking sites for a number of other cytoskeletal proteins (32), in particular vinculin (31).

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confidence: 99%

Structural Dynamics of α-Actinin-Vinculin Interactions

Bois

Borgon

Vonrhein

et al. 2005

Molecular and Cellular Biology

123

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“…For example, the ␣-actinin spectrin repeats unfold when subjected to mechanical stress (42), which occurs following the formation of adhesion complexes, allowing the exposure of new docking sites for additional cytoskeletal proteins (9). Also, the C-terminal domains of FAK and vinculin have been reported to partially unfold to allow protein-protein interactions (20).…”

Section: Discussionmentioning

confidence: 99%

The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges

Rodius

Chaloin

Moes

et al. 2008

Journal of Biological Chemistry

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Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin ␤ subunits to the cytoskeleton (Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280 -17288). Through the combined approach of mutational analysis of the ␤3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin ␤3-talin rod interaction relies on a helix-helix association between ␣-helix 50 of the talin rod domain and the membrane-proximal ␣-helix of the ␤3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin ␤3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the ␤3 subunit cytoplasmic helix as the integrin ␣IIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the ␤3 subunit following integrin activation.Integrins are ␣␤ heterodimeric receptors that mediate attachment of cells to the extracellular matrix (ECM) and therefore play important roles in cell adhesion, migration, proliferation, and survival (2, 3). Integrin clustering at sites of cellular attachment to the ECM triggers the assembly of large multifunctional submembrane protein complexes called focal adhesions (FAs), 5 that orchestrate the two-directional processing of stimuli across the cell membrane (4, 5). Both the integrin ␣ and ␤ chains participate in regulating the integrin extracellular ligand binding capacity, while the ␤ chain alone appears to link integrins to the actin cytoskeleton. Among the cytoskeletal proteins that directly interact with the ␤ chain cytoplasmic domain, four proteins: talin, ␣-actinin, filamin, and tensin possess both integrin and actin binding affinities (6, 7). Talin and ␣-actinin also bind to vinculin, an additional major component of FAs with actin binding activity.Talin and ␣-actinin are of particular interest as they have two integrin binding sites as well as multiple vinculin binding sites and display a similar structural organization comprising an extended rod domain composed of ␣-helical bundles. The ␣-actinin central rod domain, which is composed of 4 spectrin repeats of 3 ␣-helices (8, 9), associates with integrin ␤ subunits as well as vinculin through distinct helix-helix interactions (10 -16). Also, the 3 major talin rod-vinculin head (Vh) contacts have a similar architecture based on hydrophobic helixhelix interactions (17)(18)(19). Other FA proteins, such as members of the paxillin supergene family comprising paxillin, Hic-5, leupaxin, and PaxB, rely on an ␣-helical LD motif for their inter...

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“…Thus, in their resting state, the rod domains of talin or ␣-actinin have a rather low affinity for vinculin (15,20), and this has suggested a combinatorial model for vinculin activation, where simultaneous binding of two of more ligands are required to provide the free energy necessary to break the head-tail interactions of vinculin (15,18). However, this model only considers talin or ␣-actinin in their inactive states and the structure of the helical bundle domains of talin or ␣-actinin may also be dynamic as, for example, atomic force microscopy has shown that helical bundle domains that comprise spectrin repeats (also found in ␣-actinin) can form stable, unfolded intermediates when exposed to mechanical stress (44). Thus, when exposed to tension forces from within or from outside the cell, such as occurs during the formation of adhesion complexes (45)(46)(47)(48), the VBSs present in the helical bundle domains of talin and ␣-actinin might become exposed to bind to and activate vinculin, without the need for co-stimulatory signals.…”

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The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

Bois

O’Hara

Nietlispach

et al. 2006

Journal of Biological Chemistry

122

168

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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 ␣-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 ␣-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 ␣-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and ␣-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 ␣-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.Adhesion complexes form on the cell membrane when cells come in contact with each other or with components of the extracellular matrix and trigger elaborate signaling networks that direct dynamic and rapid rearrangements of the actin cytoskeleton (1, 2). Vinculin is a highly conserved and critical regulator of both cell-cell and cell-matrix junctions, as it anchors these junctions to the actin cytoskeleton by binding to talin and ␣-actinin, which directly interact with integrin and/or cadherin transmembrane receptors in focal adhesions and adherens junctions, respectively (3, 4). Overall, vinculin appears to stabilize these junctions, as vinculin overexpression augments the formation of adhesion complexes and prevents cell migration (5), whereas vinculin loss leads to marked defects in adhesion with the extracellular matrix, cell spreading, and concomitant increases in the rates of (chaotic) cell migration (6, 7). Furthermore, vinculin appears to play an important role in cancer, where it functions as a tumor suppressor that inhibits cell invasion and metastasis (8), and in other pathophysiological scenarios, including wound healing, ischemia, and apoptosis (9 -11).Vinculin is a 117-kDa modular protein that is comprised of five helical bundle domains (Vh1, Vh2, Vh3, Vt2, and Vt, Ref. 12). Intramolecular interactions of its N-terminal seven-helical bundle (Vh1) domain with its C-terminal five-helical bundle tail (Vt) domain clamp vinculin in its inactive closed conformation (12-15). Biochemical studies have shown high affinity interactions of isolated Vh1 and Vt domains (14, 16), and additional interdomain interactions of Vt with the Vt2 domain have been proposed to further ...

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Pathways and Intermediates in Forced Unfolding of Spectrin Repeats

Cited by 74 publications

References 50 publications

Structural Dynamics of α-Actinin-Vinculin Interactions

Structural Dynamics of α-Actinin-Vinculin Interactions

The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges

The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

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