Department of Chemical Engineering, ‡Centre for Condensed Matter Theory, Department of Physics, and §Centre for Biosystems Science and Engineering, Indian Institute of Science , Bengaluru, India 560012.
Langmuir. 2017 Oct 24;33(42):11496-11510. doi: 10.1021/acs.langmuir.7b02277. Epub 2017 Oct 3.
Protein membrane interactions play an important role in our understanding of diverse phenomena ranging from membrane-assisted protein aggregation to oligomerization and folding. Pore-forming toxins (PFTs) are the primary vehicle for infection by several strains of bacteria. These proteins which are expressed in a water-soluble form (monomers) bind to the target membrane and conformationally transform (protomers) and self-assemble to form a multimer transmembrane pore complex through a process of oligomerization. On the basis of the structure of the transmembrane domains, PFTs are broadly classified into β or α toxins. In contrast to β-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains has hindered our understanding of α-PFT pore formation. In this article, we use molecular dynamics (MD) simulations to examine the process of pore formation of the bacterial α-PFT, cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging from 50 to 500 ns, we show that transmembrane oligomeric intermediates or "arcs" form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the time scale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella sampling with a coarse-grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane. A single inserted protomer has a stabilization free energy of -52.9 ± 1.2 kJ/mol and forms a stable transmembrane water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore-formation pathway for ClyA.
蛋白质与膜的相互作用在我们理解从膜辅助的蛋白质聚集到寡聚化和折叠等各种现象中起着重要作用。成孔毒素（PFT）是几种细菌感染的主要媒介。这些以水溶性形式（单体）表达的蛋白质与靶膜结合，构象发生转变（原聚体）并自我组装，通过寡聚化过程形成多聚体跨膜孔复合物。根据跨膜结构域的结构，PFT 广泛分为β或α毒素。与β-PFT 相比，缺乏可用的晶体结构以及跨膜结构域的两亲性阻碍了我们对α-PFT 孔形成的理解。在本文中，我们使用分子动力学（MD）模拟来研究细菌α-PFT，即大肠杆菌细胞溶素 A（ClyA）在脂质双层膜中的孔形成过程。使用 50 至 500 ns 范围内的原子 MD 模拟，我们表明跨膜寡聚化中间体或“弧”形成稳定的脂蛋白复合物，由带有环形脂质排列的自由边缘的蛋白弧组成。通过创建脂质包含在弧内的初始条件，我们研究了自发脂质排空和环形边缘形成的动力学。这个过程发生在几十纳秒的时间尺度上，表明一旦原聚体寡聚化，跨膜弧就会迅速稳定下来，形成能够渗漏的功能性水通道。我们使用粗粒化分子模型的伞状采样来获得单个原聚体插入膜中的自由能。单个插入的原聚体具有-52.9 ± 1.2 kJ/mol 的稳定自由能，并形成能够渗漏的稳定跨膜水通道。我们的模拟表明，弧是稳定的可行中间体，可以在 ClyA 的孔形成途径中发生。
