Center for Theoretical Chemistry, Ruhr University Bochum, Bochum, Germany.
Biophys J. 2024 Aug 20;123(16):2522-2536. doi: 10.1016/j.bpj.2024.06.020. Epub 2024 Jun 21.
The ATP-binding cassette transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug-resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked," respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics (MD) simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter into the cavity of the straight P-gp conformer solved in detergent. However, access to the cavity of the kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp conformers and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between the two P-gp conformers.
三磷酸腺苷结合盒转运蛋白 P-糖蛋白（P-gp）是一种多药外排泵，在多种癌症中过度表达，并与耐药现象有关。先前已经在去污剂和纳米盘中确定了 P-gp 的结构，分别发现了不同的跨膜螺旋构象，“直”和“扭结”，这表明脂质环境对 P-gp 结构整体可能有一定的影响。在这里，我们使用模拟高度特化的肝细胞膜的不对称多组分脂质双层中的全原子分子动力学（MD）模拟，研究了两种人类 P-gp 内向开放构象体（直构象和扭结构象）的动态构象整体和蛋白-脂质相互作用，P-gp 在这种膜中表达。发现这两种构象在底物腔的可及性方面存在差异。MD 模拟表明胆固醇和不同的脂质物种如何楔入、潜入并部分进入在去污剂中解决的直构象 P-gp 腔。然而，进入在纳米盘中解决的扭结 P-gp 构象的腔受到限制。此外，两种 P-gp 构象之间的底物腔的体积和动态波动有很大差异，并受膜中胆固醇的存在（或不存在）和/或 ATP 的调节。从机制的角度来看，这些发现表明，在 P-gp 的功能工作循环中，直构象可能先于扭结构象，后者构象代表了结合底物后的状态。扭结构象中主要跨膜腔的不可及性可能在防止底物脱离和转运撤回方面至关重要。值得注意的是，在我们的无偏 MD 模拟中，直构象的一条跨膜螺旋（TM10）自发地转变为扭结构象，这强调了两种构象在天然磷脂环境中的相关性，并揭示了定义两种 P-gp 构象之间转变的结构描述符。
