Atomistic View on Substrate Transport in an ABC Exporter

ATP结合盒（ABC）输出器是ATP驱动的分子机器，其泵送广泛范围的运输底物穿过生物膜。其工作机制是基于化学机械耦合的ATP结合和水解的核苷酸结合结构域的跨膜结构域的大规模构象转变，形成易位通道的基板。然而，虽然底物转运是其关键的生物学功能，ABC出口商运输底物分子跨膜的详细机制仍然知之甚少。这种差异部分是由于表征这种内在的动态过程中的原子细节，这涉及到耦合的转运蛋白构象和基板位置的变化的挑战。两个关键方面突出作为基本上未解决的：（i）的序列的构象状态，通过它的运输周期和它们的链接到实际的基板易位过程中，和（ii）的自由能配置文件，以及它们如何比较无基板和加载的转运。因此，本项目的目的是通过全原子分子动力学（MD）模拟来表征ABC出口商中底物易位的构象动力学和能量学。异源二聚体ABC出口TM 287/288，一种细菌同源物的渗透性糖蛋白（Pgp），将作为一个原型进行研究。 首先，将进行多微秒无偏MD模拟，以研究转运底物维拉帕米的结合和TM 287/288中相关的构象变化。第二，将进行分子动力学模拟，以获得自由能景观沿着集体变量，描述与底物易位有关的TM287/288构象变化。该项目基于初步的MD模拟/EPR光谱联合工作，在没有底物的情况下，将揭示基板通过运输机和潜在的原子驱动力和机械耦合所采取的路线。此外，通过比较有和无底物的自由能分布，我们的目的是了解底物诱导的活性增强的分子基础。此外，我们将解决什么阻止底物再摄取的开放问题。考虑到近年来快速增长的X射线和冷冻EM结构的数量，这项研究的目的是提供失踪的原子级图片的结构动力学以及自由能的配置文件，是必要的理解-并可能修改-ABC转运功能。

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.