Berselli Alessandro, Alberini Giulio, Cerioni Linda, Benfenati Fabio, Maragliano Luca
Center for Synaptic Neuroscience and Technology (NSYN@UniGe), Istituto Italiano di Tecnologia, Genoa, Italy.
Department of Experimental Medicine, Università degli Studi di Genova, Genoa, Italy.
Protein Sci. 2025 Sep;34(9):e70271. doi: 10.1002/pro.70271.
In the blood-brain barrier (BBB), endothelial cells are joined by tight junctions (TJs), multi-protein assemblies that seal the paracellular space and restrict molecular transport. Among the BBB TJ proteins, Claudin-5 (Cldn15) is the most abundant one. Structural models for claudin complexes, first introduced for channel-forming, selectively permeable claudins, comprise protomers arranged to form paracellular pores that regulate transport by electrostatic and/or steric effects arising from pore-lining residues. With limited exceptions, computational studies explored oligomers of only a few subunits, while TJs are formed by extended polymeric strands. Here, we employ multi-microsecond all-atom molecular dynamics and free-energy (FE) calculations to study two distinct models of TJ-forming Cldn15 complexes, called multi-Pore I and multi-Pore II, each comprising 16 protomers arranged around three adjacent pores. FE calculations of water and ions permeation reveal that, in both models, ion transport is hindered by FE barriers higher than in single pores. Moreover, only the multi-Pore I model captures the Cldn15 G60R variant's effect, making it anion-permeable. The results provide insights into Cldn15 structure and function and validate a structural model of BBB TJs useful for studying barrier impairment in brain diseases and for developing therapeutic approaches.
在血脑屏障（BBB）中，内皮细胞通过紧密连接（TJ）相连，紧密连接是一种多蛋白组装体，可封闭细胞旁间隙并限制分子运输。在血脑屏障紧密连接蛋白中，Claudin-5（Cldn15）最为丰富。Claudin复合物的结构模型最初是针对形成通道、具有选择性通透性的Claudin提出的，该模型包括原聚体，这些原聚体排列形成细胞旁孔，通过孔壁残基产生的静电和/或空间效应来调节运输。除了少数例外情况，计算研究仅探索了几个亚基的寡聚体，而紧密连接是由延伸的聚合物链形成的。在这里，我们采用多微秒全原子分子动力学和自由能（FE）计算来研究两种不同的形成紧密连接的Cldn15复合物模型，分别称为多孔I和多孔II，每个模型都包含围绕三个相邻孔排列的16个原聚体。水和离子渗透的自由能计算表明，在这两种模型中，离子运输都受到比单孔更高的自由能屏障的阻碍。此外，只有多孔I模型捕捉到了Cldn15 G60R变体的效应，使其具有阴离子通透性。这些结果为Cldn15的结构和功能提供了见解，并验证了一种血脑屏障紧密连接的结构模型，该模型有助于研究脑部疾病中的屏障损伤以及开发治疗方法。
