Department of Chemical Engineering, Indian Institute of Science, Bangalore, India.
Department of Chemical Engineering, and Center for BioSystems Science and Engineering, Indian Institute of Science, Bangalore, India.
J Membr Biol. 2020 Dec;253(6):535-550. doi: 10.1007/s00232-020-00148-9. Epub 2020 Oct 29.
Pore-forming toxins are proteins expressed by bacteria to primarily cause infections in the host cell. Cholesterol-dependent cytolysins (CDCs) are a class of proteins whose pore-forming ability requires the presence of cholesterol in the membrane. Upon binding to the target cell, cholesterol-recognizing residues in the membrane binding D4 subdomain assist in stabilizing both the pre-pore and pore states which occur during protein oligomerization on the cell membrane. Super resolution-stimulated emission depletion (STED) microscopy experiments (Sarangi et al. in Langmuir, 32:9649-9657, 2016) on supported lipid bilayers have shown that listeriolysin (LLO), a CDC expressed by Listeria monocytogenes, a food-borne pathogen, induces both spatial and dynamic heterogeneity in bilayer membranes. Here, we use all-atom molecular dynamics simulations to explore molecular details of the induced membrane reorganization by considering two distinct states of the oligomerized LLO protein in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): cholesterol membrane. In the membrane bound (MB) state, four D4 subunits are placed at the bilayer interface in a pre-pore configuration and the membrane-inserted (MI) state consists of a tetrameric arc-like pore configuration. By analyzing lipid-order parameters, mobilities, and diffusion coefficients, we examine the induced spatial heterogeneity that occurs in both the MB and MI states. This heterogeneity is primarily driven by the local density enhancement of cholesterol in the vicinity of the MB, D4 subunits leading to distinct differences in lipid and cholesterol mobility across the two leaflets as well as enhanced lipid mobilities in regions where cholesterol is depleted. The leaflet-induced heterogeneity is greater for the MB state when compared with the MI state and the dynamic variations are more pronounced in the extracellular leaflet when compared with the cytosolic leaflet. Our study provides molecular-level insights into the inhomogeneity and perturbation induced in bilayer membranes upon LLO binding and pore formation and is expected to represent trends across PFTs in the broad CDC subclass of proteins.
孔形成毒素是细菌表达的蛋白质，主要引起宿主细胞感染。胆固醇依赖性细胞溶解素（CDCs）是一类蛋白质，其孔形成能力需要膜中的胆固醇存在。在与靶细胞结合后，膜结合 D4 亚域中的胆固醇识别残基有助于稳定前孔和孔状态，这些状态发生在细胞膜上的蛋白质寡聚化过程中。受激发射损耗（STED）显微镜实验（Sarangi 等人，在 Langmuir，32：9649-9657，2016 年）在支撑脂质双层上表明，李斯特菌溶血素（LLO），一种由李斯特菌属表达的 CDC，是一种食源性病原体，诱导双层膜中的空间和动态异质性。在这里，我们使用全原子分子动力学模拟来探索通过考虑寡聚化 LLO 蛋白的两种不同状态在 1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）：胆固醇膜中的分子细节来诱导的膜重排。在膜结合（MB）状态下，四个 D4 亚基位于双层界面的前孔构象中，而膜插入（MI）状态由四聚体弧形孔构象组成。通过分析脂质有序参数、迁移率和扩散系数，我们研究了在 MB 和 MI 状态下发生的诱导空间异质性。这种异质性主要由 MB 附近胆固醇的局部密度增强引起，导致两个叶状层之间的脂质和胆固醇迁移率以及胆固醇耗尽区域的脂质迁移率存在明显差异。与 MI 状态相比，MB 状态下的叶状层诱导异质性更大，与细胞质层相比，细胞外叶层的动态变化更为明显。我们的研究提供了分子水平的见解，即LLO 结合和孔形成后双层膜中的非均质性和扰动，并有望代表广泛的 CDC 亚类蛋白质中的 PFT 趋势。
