Distinct autoinhibitory mechanisms regulate vinculin binding by αT-catenin and αE-catenin

ABSTRACTα-catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of αT(testes)-catenin, an α-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that αT-catenin binds the β-catenin/N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.

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Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin.

The Journal of Cell Biology ◽

10.1083/jcb.102.4.1439 ◽

1986 ◽

Vol 102 (4) ◽

pp. 1439-1446 ◽

Cited By ~ 54

Author(s):

J Bryan ◽

S Hwo

Keyword(s):

Binding Site ◽

Limited Proteolysis ◽

Gel Filtration ◽

Actin Binding ◽

Binding Studies ◽

Terminal Fragment ◽

Actin Binding Domain ◽

Nucleation Activity ◽

Carboxyl Terminal ◽

Definition Of

Brevin is a Ca2+-modulated actin-associated protein that will sever F-actin and cap barbed filament ends. Limited proteolysis with chymotrypsin or subtilisin cleaves the molecule approximately in half. Cleavage is approximately 10-fold more rapid in Ca2+ than in EGTA. The two fragments are readily separated from each other and from undigested brevin by high pressure liquid chromatography on a DEAE resin. A 40,000-mol-wt fragment from the N-terminal is not retained by DEAE, while a 45,000-mol-wt C-terminal fragment binds more tightly than brevin. The N-terminal fragment retains approximately 10% of the nucleation activity, caps barbed ends, and retains 50% of the total severing activity defined by dilution induced depolymerization of pyrenyl actin, but, in contrast to brevin, none of these functions are affected by Ca2+. Fluorescent actin binding studies and gel-filtration demonstrate that the 40,000-mol-wt fragment binds two actin monomers. The 45,000-mol-wt C-terminal fragment has no severing, nucleating, or capping activity. Cross-reaction with two monoclonal antibodies against two specific Ca2+-induced conformations of human platelet gelsolin suggest that both Ca2+ binding sites are located on the carboxyl half of the brevin molecule. One epitope, defined as the rapidly exchanging Ca2+ binding site in the gelsolin-actin complex, is lost when a 20,000-mol-wt fragment is cleaved from the carboxyl terminal. The second epitope, related to the poorly exchanging Ca2+ binding site in the complex, is nearer the middle of the brevin molecule.

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Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling

Scientific Reports ◽

10.1038/s41598-019-51816-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Ksenia Terekhova ◽

Sabine Pokutta ◽

Yee S. Kee ◽

Jing Li ◽

Emad Tajkhorshid ◽

...

Keyword(s):

Conformational Changes ◽

Binding Protein ◽

Binding Domain ◽

Actin Binding ◽

Titration Calorimetry ◽

Allosteric Coupling ◽

Actin Binding Protein ◽

Binding Partners ◽

Actin Binding Domain ◽

Cysteine Labeling

Abstract Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.

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Espin Contains an Additional Actin-binding Site in Its N Terminus and Is a Major Actin-bundling Protein of the Sertoli Cell–Spermatid Ectoplasmic Specialization Junctional Plaque

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4327 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4327-4339 ◽

Cited By ~ 50

Author(s):

Bin Chen ◽

Anli Li ◽

Dennis Wang ◽

Min Wang ◽

Lili Zheng ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Sertoli Cell ◽

Actin Binding ◽

Actin Stress Fiber ◽

Splice Isoforms ◽

Actin Bundles ◽

N Terminus ◽

Ectoplasmic Specialization ◽

Actin Binding Site

The espins are actin-binding and -bundling proteins localized to parallel actin bundles. The 837-amino-acid “espin” of Sertoli cell–spermatid junctions (ectoplasmic specializations) and the 253-amino-acid “small espin” of brush border microvilli are splice isoforms that share a C-terminal 116-amino-acid actin-bundling module but contain different N termini. To investigate the roles of espin and its extended N terminus, we examined the actin-binding and -bundling properties of espin constructs and the stoichiometry and developmental accumulation of espin within the ectoplasmic specialization. An espin construct bound to F-actin with an approximately threefold higher affinity (K d = ∼70 nM) than small espin and was ∼2.5 times more efficient at forming bundles. The increased affinity appeared to be due to an additional actin-binding site in the N terminus of espin. This additional actin-binding site bound to F-actin with a K d of ∼1 μM, decorated actin stress fiber-like structures in transfected cells, and was mapped to a peptide between the two proline-rich peptides in the N terminus of espin. Espin was detected at ∼4–5 × 106 copies per ectoplasmic specialization, or ∼1 espin per 20 actin monomers and accumulated there coincident with the formation of parallel actin bundles during spermiogenesis. These results suggest that espin is a major actin-bundling protein of the Sertoli cell–spermatid ectoplasmic specialization.

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Isolation of an actin-binding fragment of fibronectin

Biochemical Journal ◽

10.1042/bj1930615 ◽

1981 ◽

Vol 193 (2) ◽

pp. 615-620 ◽

Cited By ~ 49

Author(s):

J Keski-Oja ◽

K M Yamada

Keyword(s):

Affinity Chromatography ◽

Binding Site ◽

Limited Proteolysis ◽

Actin Binding ◽

Collagen Binding ◽

Actin Binding Site

We have identified a specific actin-binding site in the adhesive glycoprotein fibronectin, isolated from chicken fibroblasts. Affinity chromatography of fragments, released from fibronectin by limited proteolysis with trypsin, chymotrypsin and subtilisin, on actin-Sepharose and other protein-Sepharose columns was used to locate the binding site. A 27 000-mol.wt. subtilisin-digest fragment bound efficiently to actin. The results suggest that the actin-binding site is close to, but not identical with, the reported collagen-binding site.

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Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface

Biochemical Journal ◽

10.1042/bj20040875 ◽

2005 ◽

Vol 385 (3) ◽

pp. 659-665 ◽

Cited By ~ 4

Author(s):

Ulrike FOCK ◽

Brigitte M. JOCKUSCH ◽

Wolf-Dieter SCHUBERT ◽

Horst HINSSEN

Keyword(s):

Tertiary Structure ◽

Limited Proteolysis ◽

Cell Types ◽

Actin Binding ◽

Plasma Gelsolin ◽

Equine Plasma ◽

Binding Epitope ◽

N Terminus ◽

Immunological Approach ◽

Cytoplasmic Gelsolin

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34–Ile45 and Leu64–Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

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