Theoretical Chemistry, Faculty of Chemistry and Biochemistry , Ruhr-University Bochum , 44780 Bochum , Germany.
EPR Spectroscopy, Faculty of Chemistry and Biochemistry , Ruhr-University Bochum , 44780 Bochum , Germany.
J Am Chem Soc. 2018 Apr 4;140(13):4543-4551. doi: 10.1021/jacs.7b12944. Epub 2018 Mar 26.
ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously.
ATP 结合盒（ABC）转运蛋白是 ATP 驱动的分子机器，其中核苷酸结合域（NBD）中的 ATP 结合和水解与跨膜域（TMD）中的大规模交替访问构象变化在化学机械上偶联，最终导致底物跨生物膜的转运。大尺度构象转变背后的结构动力学的精确性质以及 NBD 和 TMD 运动的偶联仍然没有解决。在这项工作中，我们将全原子分子动力学（MD）模拟与电子顺磁共振（EPR）光谱相结合，以揭示异二聚体 ABC 外排泵 TM287/288 功能工作循环基础上的动态构象转变的原子水平机制。在明确的膜/水环境中进行的广泛的多微秒模拟表明，TM287/288 如何在 ATP 结合后自发地从内向（IF）状态通过被阻塞（Occ）中间体过渡到外向（OF）状态。迄今为止，后两种状态尚未在原子水平上进行表征。ATP 诱导的 NBD 二聚体的收紧涉及两个 NBD 单体的闭合和重定向，同时伴随细胞内 TMD 门的闭合，导致被阻塞状态。随后，细胞外 TMD 门的打开产生 OF 构象。所获得的机制通过在两个域之间以及 TMD 内的构象转变之间进行紧密的协调来施加 NBD-TMD 偶联，确保细胞质和周质门区域永远不会同时打开。
