Atomistic View on Substrate Transport in an ABC Exporter

三磷酸腺苷结合盒(ABC)出口器是一种由三磷酸腺苷驱动的分子机器，可以通过生物膜泵送广泛的运输底物。它们的作用机制是基于核苷酸结合区的ATP结合和水解与形成底物易位通道的跨膜区的大规模构象转变的化学机械耦合。然而，尽管底物转运是它们的关键生物学功能，但ABC出口蛋白转运底物分子的详细机制仍然知之甚少。这种差异的部分原因是在原子细节上表征这一内在动态过程的挑战，这涉及转运蛋白构象和底物位置的耦合变化。有两个关键方面基本上尚未解决：(1)转运蛋白循环的构象状态序列及其与实际底物转运过程的联系；(2)无底物转运蛋白和有底物转运蛋白的自由能分布及其比较情况。因此，本项目旨在通过全原子分子动力学(MD)模拟来表征ABC输出子中底物转运的构象动力学和能量学。异二聚体ABC Exporter TM287/288是通透性糖蛋白(Pgp)的细菌同源物，将作为原型进行研究。首先，将进行多微秒无偏MD模拟来研究转运底物维拉帕米的结合以及TM287/288中相关的构象变化。其次，将进行元动力学模拟以获得描述TM287/288与底物移位相关的构象变化的集体变量的自由能景观。该项目基于在没有底物的情况下对TM287/288进行的初步MD模拟/EPR光谱工作，将揭示底物通过转运体的路径以及潜在的原子驱动力和机械耦合。此外，通过比较有无底物的自由能分布，我们旨在了解底物诱导活性增强的分子基础。此外，我们还将解决一个悬而未决的问题，即是什么阻止底物再吸收。考虑到近年来X射线和低温EM结构的数量迅速增加，这项拟议的研究旨在提供结构动力学的缺失原子水平图像以及理解-并可能修改-ABC转运体函数所必需的自由能分布。

ATP-binding cassette (ABC) exporters are ATP-driven molecular machines that pump a broad range of transport substrates across biological membranes. Their working mechanism is based on chemomechanical coupling of ATP binding and hydrolysis in the nucleotide-binding domains to large-scale conformational transitions of the transmembrane domains that form the translocation channel for the substrate. However, although substrate translocation is their key biological function, the detailed mechanism by which ABC exporters transport substrate molecules across membranes is still poorly understood. This discrepancy is partly due to the challenges of characterizing this intrinsically dynamic process in atomic detail, which involves coupled changes of transporter conformation and substrate position. Two key aspects stand out as essentially unresolved: (i) The sequence of conformational states through which the transporter cycles and their link to the actual substrate translocation process, and (ii) the free energy profiles and how they compare for substrate-free and -loaded transporters. This project therefore aims at characterizing the conformational dynamics and energetics of substrate translocation in an ABC exporter by all-atom molecular dynamics (MD) simulations. The heterodimeric ABC exporter TM287/288, a bacterial homolog of permeability glycoprotein (Pgp), will be investigated as a prototype. First, multi-microsecond unbiased MD simulations will be carried out to investigate binding of transport substrate verapamil and the associated conformational changes in TM287/288. Second, metadynamics simulations will be carried out to obtain free energy landscapes along collective variables that describe the conformational changes of TM287/288 linked to substrate translocation.This project, which is based on preliminary combined MD simulation/EPR spectroscopy work on TM287/288 in absence of substrate, will reveal the route taken by the substrate through the transporter and the underlying atomic driving forces and mechanical couplings. Furthermore, by comparing the free energy profiles with and without substrate, we aim at understanding the molecular basis for substrate-induced activity enhancement. In addition, we will tackle the open question of what prevents substrate reuptake. Considering the rapidly growing number of X-ray and cryo-EM structures in recent years, this proposed study aims at providing the missing atomic-level picture of the structural dynamics as well as free energy profiles that are necessary to understand – and possibly modify – ABC transporter function.