Distinct intramolecular interactions regulate autoinhibition of vinculin binding in αT-catenin and αE-catenin
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100582
ISSN1083-351X
AutoresJonathon A. Heier, Sabine Pokutta, Ian W. Dale, Sun Kyung Kim, Andrew P. Hinck, William I. Weis, Adam V. Kwiatkowski,
Tópico(s)Force Microscopy Techniques and Applications
Resumoα-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. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry 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 the 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 show 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. α-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. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry 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. 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Despite a growing awareness of its importance in human disease, the molecular properties and ligand interactions of αT-catenin remain poorly understood. Our previous work revealed that αT-catenin, unlike mammalian αE-catenin, is a monomer in the solution that can bind to F-actin with low micromolar affinity in the absence of tension. F-actin binding is also not allosterically regulated, as the β-catenin–αT-catenin complex binds to F-actin with the same affinity as the αT-catenin monomer (38Wickline E.D. Dale I.W. Merkel C.D. Heier J.A. Stolz D.B. Kwiatkowski A.V. AlphaT-catenin is a constitutive actin-binding alpha-catenin that directly couples the Cadherin.Catenin complex to actin filaments.J. Biol. Chem. 2016; 291: 15687-15699Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Single-molecule pulling experiments have shown the αT-catenin M-region to be mechanoresponsive as it unfurls in a force range similar to αE-catenin (47Pang S.M. Le S. Kwiatkowski A.V. Yan J. 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We measured the recruitment of vinculin to cell–cell contacts and found that, despite the distinct mechanism of regulation, recruitment of vinculin is still tension dependent. Our findings indicate that the way in which tension regulates vinculin binding differs between αE-catenin and αT-catenin. We postulate that this mechanism is important for the ability of αT-catenin to build specific and distinct molecular complexes at AJs. We characterized the interaction between αT-catenin and β-catenin by isothermal titration calorimetry (ITC) using purified recombinant proteins. We used the head region (comprising the N- and M-domains) of αT-catenin (aa 1–659, Fig. 1A) for these experiments because it is more stable than full-length αT-catenin and yields sufficiently high protein concentrations for ITC. Past studies revealed that the αE-catenin head region (aa 1–651) binds β-catenin and the β-catenin–E-cadherin tail complex with a similar affinity to full-length αE-catenin (12Pokutta S. Choi H.J. Ahlsen G. Hansen S.D. Weis W.I. Structural and thermodynamic characterization of cadherin.beta-catenin.alpha-catenin complex formation.J. Biol. Chem. 2014; 289: 13589-13601Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We observed that the αT-catenin head binds β-catenin with a dissociation constant ∼250 nM (Fig. 1B; Table 1). The affinity of αT-catenin for β-catenin is an order of magnitude weaker than the association of αE-catenin or αN-catenin for β-catenin (15–20 nM; (12Pokutta S. Choi H.J. Ahlsen G. Hansen S.D. Weis W.I. Structural and thermodynamic characterization of cadherin.beta-catenin.alpha-catenin complex formation.J. Biol. Chem. 2014; 289: 13589-13601Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar)).Table 1ITC measurements of αT-catenin fragments binding to β-catenin or β-catenin–N-cadherin cytoplasmic tail complexProteinsKd (nM)ΔH (kcal/mol)TΔS (kcal/mol)ΔG (kcal/mol)#αT-catenin head (1–659) + β-catenin264.1 ± 109.1−14.6 ± 2.1−5.6−9.05αT-catenin head (1–659) + β-catenin–Ncadcyto5.6 ± 0.6−27.0 ± 0.2−15.8−11.33αT-catenin N1-N2 (1–259) + β-catenin–Ncadcyto6.9 ± 12.0−18.9 ± 1.0−7.8−11.11Ncadcyto, N-cadherin tail. Open table in a new tab Ncadcyto, N-cadherin tail. Cadherin tail binding to β-catenin strengthens the affinity between β-catenin and α-catenin (12Pokutta S. Choi H.J. Ahlsen G. Hansen S.D. Weis W.I. Structural and thermodynamic characterization of cadherin.beta-catenin.alpha-catenin complex formation.J. Biol. Chem. 2014; 289: 13589-13601Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). N-cadherin is the primary classical cadherin expressed in cardiomyocytes (48Kostetskii I. Li J. Xiong Y. Zhou R. Ferrari V.A. Patel V.V. Molkentin J.D. Radice G.L. Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure.Circ. Res. 2005; 96: 346-354Crossref PubMed Scopus (244) Google Scholar). We tested if the N-cadherin tail (Ncadcyto) influences the αT-catenin–β-catenin interaction by titrating the β-catenin–Ncadcyto complex into αT-catenin head (Fig. 1C). The affinity of this interaction was 5 to 6 nM (Table 1), indicating that αT-catenin binds to the cadherin–β-catenin complex an order of magnitude more strongly than to β-catenin alone. This affinity is similar to the 1- to 2-nM affinity observed between the cadherin tail–β-catenin complex and αE-catenin or αN-catenin (12Pokutta S. Choi H.J. Ahlsen G. Hansen S.D. Weis W.I. Structural and thermodynamic characterization of cadherin.beta-catenin.alpha-catenin complex formation.J. Biol. Chem. 2014; 289: 13589-13601Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and suggests that αT-catenin can effectively compete with αE-catenin for binding to the membrane-associated cadherin–β-catenin complex. Full-length αT-catenin is primarily a monomer in the solution, although it does have homodimerization potential in vitro (38Wickline E.D. Dale I.W. Merkel C.D. Heier J.A. Stolz D.B. Kwiatkowski A.V. AlphaT-catenin is a constitutive actin-binding alpha-catenin that directly couples the Cadherin.Catenin complex to actin filaments.J. Biol. Chem. 2016; 291: 15687-15699Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The best evidence for dimerization potential comes from a point mutation linked to arrhythmogenic ventricular cardiomyopathy in humans, V94D, that renders αT-catenin an obligate homodimer (38Wickline E.D. Dale I.W. Merkel C.D. Heier J.A. Stolz D.B. Kwiatkowski A.V. AlphaT-catenin is a constitutive actin-binding alpha-catenin that directly couples the Cadherin.Catenin complex to actin filaments.J. Biol. Chem. 2016; 291: 15687-15699Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 44van Hengel J. Calore M. Bauce B. Dazzo E. Mazzotti E. De Bortoli M. Lorenzon A. Li Mura I.E. Beffagna G. Rigato I. Vleeschouwers M. Tyberghein K. Hulpiau P. van Hamme E. Zaglia T. et al.Mutations in the area composita protein alphaT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy.Eur. Heart J. 2013; 34: 201-210Crossref PubMed Scopus (123) Google Scholar). We analyzed the oligomerization properties of the αT-catenin N1-N2 (aa 1–259, Fig. 1A) and compared them to the V94D mutant. Analytical size-exclusion chromatography of αT-catenin WT N1-N2 and V94D N1-N2 revealed that WT N1-N2 eluted as a single monomer species (Fig. 2A, blue line), whereas the V94D mutant eluted as a dimer species (Fig. 2A, red line). We then analyzed the oligomeric state of the αT-catenin N-terminus by cross-linking. Increasing concentrations of αT-catenin N1-N2 were incubated with or without the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at 4 °C or 37 °C and the resulting products analyzed by SDS-PAGE. As expected, the αT-catenin N1-N2 migrated as a 25-kDa protein in the absence of a cross-linker (Fig. 2B). Incubation with BS3 did not affect migration at low concentrations, although at higher concentrations (4 and 8 μM), larger species were detected at both temperatures. In contrast, αT-catenin V94D N1-N2 ran as 50-kDa protein in the presence of BS3 at all concentrations tested (Fig. 2C), indicating a cross-linked dimer. We conclude that the αT-catenin N terminus, similar to full-length protein, is primarily a monomer in the solution. We then teste
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