Assembly of Tight Junction Strands: Claudin-10b and Claudin-3 Form Homo-Tetrameric Building Blocks that Polymerise in a Channel-Independent Manner
Assembly of Tight Junction Strands: Claudin-10b and Claudin-3 Form Homo-Tetrameric Building Blocks that Polymerise in a Channel-Independent Manner
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DOI：
10.1016/j.jmb.2020.02.034
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发表时间：
2020-03-27
影响因子：
5.6
通讯作者：
Piontek, J.
中科院分区：
文献类型：
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作者：
Hempel, C.;Protze, J.;Piontek, J.
紧密连接选择性地调节细胞旁通透性大小和电荷。已经提出了紧密连接链和细胞旁通道的分子结构模型。然而，它们与实验和结构数据并不完全一致。在这里，我们通过链的细胞重建、结构引导诱变、计算机蛋白质对接和寡聚物建模来分析基于密蛋白的紧密连接链和通道的结构。分析了原型通道（Cldn10b）和屏障形成（Cldn3）紧密蛋白。福斯特共振能量转移（FRET）测定表明多步密蛋白聚合，从密蛋白亚型特异性的顺式寡聚开始，然后是反式相互作用触发的顺式聚合。替代原体界面在计算机中建模，并通过半胱氨酸介导的交联、共焦和基于冷冻断裂电镜的链形成分析进行测试。分析的密蛋白突变体还包括引起 HELIX 综合征的突变。结果表明 Cldn10b 和 Cldn3 链中的原聚体形成相似的反平行双排，正如 Cldn15 所建议的那样。鉴定出相互稳定的亲水性和疏水性顺式和反式界面，其中包含细胞外片段 ECS1 和 ECS2 的新关键残基。柔性 ECS1 β 1 β 2 环的疏水聚类与 ECS2-ECS2 反式相互作用被认为是四聚体构建块连接成密蛋白聚合物的驱动力。 Cldn10b 和 Cldn3 具有相同的聚合机制。然而，在四聚体的细胞旁中心，静电排斥可能导致孔（Cldn10b）的形成和对屏障（Cldn3）的静电吸引。结合体外数据和计算机模拟，本研究通过阐明密蛋白组装及其在 HELIX 综合征中的病理改变，提高了对细胞旁通透性调节机制的理解。 (C) 2020 Elsevier Ltd. 保留所有权利。
Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Forster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising -hydrophilic and hydrophobic - cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2.Hydrophobic clustering of the flexible ECS1 beta 1 beta 2 loops together with ECS2-ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b and Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome. (C) 2020 Elsevier Ltd. All rights reserved.