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Distinct autoinhibitory mechanisms regulate vinculin binding by alpha-T-catenin and alpha-E-catenin
bioRxiv - Biochemistry Pub Date : 2020-10-26 , DOI: 10.1101/2020.10.25.354415
Jonathon A. Heier , Sabine Pokutta , Ian W. Dale , Sun Kyung Kim , Andrew P. Hinck , William I. Weis , Adam V. Kwiatkowski

Alpha-catenin binds directly to beta-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates alpha-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 alpha-T(testes)-catenin, an alpha-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that alpha-T-catenin binds the beta-catenin/N-cadherin complex with a similar low nanomolar affinity to that of alpha-E-catenin. Limited proteolysis revealed that the alpha-T-catenin M-region adopts a more open conformation than alpha-E-catenin. The alpha-T-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated alpha-T-catenin M-region is not autoinhibited and thereby distinct from alpha-E-catenin. However, the alpha-T-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, alpha-T-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 alpha-T-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of alpha-T-catenin allow it to function as a scaffold for building specific adhesion complexes.

中文翻译:

不同的自抑制机制通过alpha-T-catenin和alpha-E-catenin调节纽蛋白结合

α-catenin直接与β-catenin结合,并将钙粘蛋白-catenin复合物与肌动蛋白细胞骨架相连。张力调节α-连环蛋白的构象:肌动球蛋白产生的力使中间(M)区伸展,以缓解自身抑制作用,并揭示肌动蛋白结合蛋白纽蛋白的结合位点。在这里,我们描述了α-T(睾丸)-连环蛋白(一种对心脏功能至关重要的α-连环蛋白同工型)的生化特性,以及分子内相互作用如何调节纽蛋白结合自抑制。等温滴定热法(ITC)表明,α-T-连环蛋白以与α-E-连环蛋白相似的低纳摩尔亲和力结合β-连环蛋白/ N-钙粘着蛋白复合物。有限的蛋白水解显示,α-T-cateninM区比α-E-catenin具有更开放的构象。α-T-连环蛋白M区以低纳摩尔亲和力结合纽扣蛋白N端,表明分离的α-T-连环蛋白M区没有被自动抑制,因此不同于α-E-连环蛋白。但是,α-T-catenin头(N和M区)与新蛋白的结合弱了1000倍(低微摩尔亲和力),表明N端调节M区与新蛋白的结合。在细胞中,新蛋白的α-T-连环素募集到细胞间接触需要肌动蛋白结合域和肌动球蛋白产生的张力,这表明力调节了新蛋白的结合。在一起,我们的结果表明,需要α-T-连环蛋白N末端来维持M区域的自抑制并调节纽蛋白结合。
更新日期:2020-10-26
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