Structural and Functional Analysis of the Actin Binding Domain of Plectin Suggests Alternative Mechanisms for Binding to F-Actin and Integrin β4

García‐Álvarez, Begoña; Bobkov, Andrey A.; Sonnenberg, Arnoud; Pereda, José M. de

doi:10.1016/s0969-2126(03)00090-x

Cited by 93 publications

(118 citation statements)

References 62 publications

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“…Proteins were expressed in Escherichia coli strain BL21(DE3) and were purified by nickel-chelating affinity chromatography as described (22). The His tag present at the N terminus of the fusion proteins was cleaved by digestion with tobacco etch virus protease, and was removed by a second nickel-affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…Titration of Thiol Groups-Sulfhydryl groups were titrated under denaturing conditions with 5,5Ј-dithio-nis(2-nitrobenzoic acid) as described (22).…”

Section: Methodsmentioning

confidence: 99%

“…Near the N terminus plectin contains an actin binding domain (ABD) built up of a tandem pair of calponin homology domains (CH1 and CH2), similar to the ABDs present in dystrophin and other members of the spectrin superfamily. The ABD binds to the first pair of fibronectin type III repeats of the integrin ␤4 subunit via the CH1 domain (18 -20); the ABD also binds to F-actin (21)(22), nesprin-3␣ (11), and to nonfilamentous vimentin (23). Adjacent to the ABD extends a ϳ1000 residue long region named the plakin domain that is conserved in all plakins, except in epiplakin.…”

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confidence: 99%

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The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

Ortega

Buey

Sonnenberg

et al. 2011

Journal of Biological Chemistry

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Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudobinding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.Plakins are a family of high molecular weight proteins that interconnect elements of the cytoskeleton and tether them to membrane-associated structures; hence, they are also known as cytolinkers (1-2). Mammalian plakins include desmoplakin, plectin, the bullous pemphigoid antigen 1 (BPAG1), 4 the microtubule actin crosslinking factor 1 (MACF1, also known as ACF7, trabeculin, or macrophin), envoplakin, periplakin, and epiplakin. Plakins are also present in invertebrates; Drosophila melanogaster has a single plakin gene named shortstop (also know as kakapo) that encodes at least two protein forms, Shot I and Shot II. Similarly, the only plakin gene in Caenorhabditis elegans, vab-10, encodes two variants VAB-10A and VAB-10B.Plectin is a highly versatile plakin that associates with intermediate filaments (3), microtubules (4), and actin fibers (5), and crosslinks these cytoskeletal networks (5-6). Plectin also connects intermediate filaments to membrane-associated complexes. In stratified epithelia such as the skin, plectin is localized at the hemidesmosomes, which are junctional complexes that link the intermediate filaments to the basement membrane, and links the integrin ␣6␤4 and the bullous pemphigoid antigen 2 (BPAG2, also known as type XVII collagen or BP180) to the cytokeratins (7-8). Plectin is also localized at the desmosomes (9), which mediate cell-cell contacts, and connects the nuclear envelope to the intermediate filaments by binding to the outer nuclear membrane protein nesprin-3␣ (10 -11). In st...

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Section: Methodsmentioning

confidence: 99%

“…Titration of Thiol Groups-Sulfhydryl groups were titrated under denaturing conditions with 5,5Ј-dithio-nis(2-nitrobenzoic acid) as described (22).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

Ortega

Buey

Sonnenberg

et al. 2011

Journal of Biological Chemistry

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“…The latter remains a viable option, since integrins are known to be flexible (55), and shear may lead to an extension similar to the extended chain conformation observed for von Willebrand factor (56). It is worth noting that the integrin binding partners talin and paxillin that regulate cell adhesion, migration, and integrin conformation (57)(58)(59)(60)(61)(62)(63) could provide a means of mechanotransduction. That signaling has been documented with a magnetic drag force (64) to extend integrin molecules, generating an intracellular calcium response, gene transcription (65) and tyrosine phosphorylation (66 -68).…”

Section: Vla-4 Affinity Modulation By Shearmentioning

confidence: 99%

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Zwartz¹,

Chigaev²,

Dwyer

et al. 2004

Journal of Biological Chemistry

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Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the ␣ 4 ␤ 1 integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca 2؉ responses. Ca Leukocytes are recruited to endothelial cells in a multistep process using selectin and integrin adhesion molecules (1, 2). These molecules allow a cell to tether, roll, adhere, and transmigrate along and across an endothelial layer. Selectin and some integrin molecules and their associated ligands mediate tethering and rolling interactions. Firm adhesion is mediated by vascular ligands of the immunoglobulin superfamily such as vascular cell adhesion molecule 1 (VCAM-1) 1 and their associated integrins (1, 2). The adhesive strength or avidity (3) of cells expressing integrins can be rapidly modulated by chemokines and chemoattractants, which also regulate leukocyte recruitment and migration across vascular endothelium. The rapid changes in avidity have been attributed to changes in the number of interacting molecules or valency due to molecular redistribution or clustering and to changes in the affinity of the individual receptor-ligand bonds (3-10).Physiological shear can also regulate leukocyte traffic by stimulating mechanosensors on neutrophils, monocytes, lymphocytes, erythrocytes, and platelets (see Ref. 11 and references therein). Shear arises from bifurcating blood vessels or rapid changes in blood vessel diameters. Shear acting on leukocytes, bound to endothelial cells, produces mechanical stress on the cells or their receptors, regulating cell growth and proliferation, protein synthesis, gene expression, and blood cell recruitment (12, 13). Integrins (such as ␣ v ␤ 3 , ␣ 5 ␤ 1 , ␣ 4 ␤ 1 , and ␣ 2 ␤ 1 ) on endothelial cells can act as mechanosensors to changes in blood flow (13,14) and trigger an intracellular signaling pathway involving focal adhesion kinase and mitogen-activated protein kinase cascades. How shear specifically induces blood cell adhesiveness or recruitment through mechanosensors is unknown. Indirect evidence shows that increased integrin binding to the extracellular matrix occurs when shear acts on cells or their mechanosensors to induce intracellular signaling. For example, intracellular signaling leads to conformational changes and activation of ␣ v ␤ 3 on endothelial cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 integrins on monocytic cells (15, 16). Shear acting on endothelial cells affects the GTPase Rho signaling pathway and in monocyti...

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“…human proteins; fimbrin, 9 utrophin, 10 dystrophin 11 and plectin. 12 In the crystal form of the fimbrin and plectin ABDs, the constituent paired CH1 and CH2 domains have a closed structure with intra molecular contacts between CH domains. In contrast, CH1 and CH2 in utrophin and dystrophin ABDs are in an extended, open configuration but form dimers in the crystals, where the interactions between CH domains involve separate polypeptide chains.…”

mentioning

confidence: 99%

A 3-D Reconstruction of Smooth Muscle α-Actinin by CryoEm Reveals Two Different Conformations at the Actin-binding Region

Taylor

2004

Journal of Molecular Biology

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Structural and Functional Analysis of the Actin Binding Domain of Plectin Suggests Alternative Mechanisms for Binding to F-Actin and Integrin β4

Cited by 93 publications

References 62 publications

The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

A 3-D Reconstruction of Smooth Muscle α-Actinin by CryoEm Reveals Two Different Conformations at the Actin-binding Region

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