Complete mapping of substrate translocation highlights the role of LeuT N-terminal segment in regulating transport cycle.
Complete mapping of substrate translocation highlights the role of LeuT N-terminal segment in regulating transport cycle.
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DOI：
10.1371/journal.pcbi.1003879
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发表时间：
2014-10
影响因子：
4.3
通讯作者：
Bahar I
中科院分区：
文献类型：
--
作者：
Cheng MH;Bahar I
神经递质：钠同向转运体（NSS）通过在钠离子的共转运的辅助下从突触清除过量的神经递质来调节神经元信号传递。近年来，NSS家族的几个成员已经收集了大量的结构数据，这为基于结构的研究开辟了道路，以了解底物转运的机理。亮氨酸转运蛋白（LeuT），细菌的直系同源物，已被广泛采用作为原型在这些研究中。然而，由于全球和局部事件的复杂相互作用以及缺少LeuT N-末端片段的结构数据，这一目标一直难以实现。我们在这里首次提供了一个全面的描述的分子事件，导致基板/Na+释放到突触后细胞，包括结构和动态的N-末端段使用分子模拟的组合。底物和Na+释放遵循水分子流入底物/Na+结合口袋，伴随着跨膜螺旋的协同重排。盐桥和阳离子-π相互作用在N-末端区段的重新分布促进底物释放。重要的是，底物释放之后是细胞内门的关闭和全局重新配置回到面向外的状态以恢复运输循环。两个最低限度的水合中间体，没有结构上解决的日期，确定：一个，基板绑定，稳定的通道期间，从外向内面向状态（全息闭塞），和另一个，基板免费，沿着反向过渡（apo闭塞）。细菌亮氨酸转运体（LeuT）属于神经递质钠同向转运体（NSS）家族。它的人类直系同源物包括多巴胺转运蛋白和血清素转运蛋白。NSS成员的功能障碍与神经系统疾病有关，因此阐明其作为临床相关药物靶点的作用机制具有重要意义。NSS通过交替进入细胞外和细胞内介质共转运底物（神经递质或氨基酸）和钠离子穿过细胞膜，这使得底物和离子能够从细胞外区域摄取并释放到细胞内区域。尽管在阐明NSS家族成员的结构和功能方面取得了重大进展，但其功能机制及其暴露于细胞内部的N-末端片段的作用仍然难以捉摸。在这里，我们提供了第一次全原子时间分辨的描述LeuT的完整的运输周期，使用多尺度模拟。两个主要发现是（i）阐明了N-末端片段的结构和动力学，这有助于介导底物和阳离子释放并恢复转运循环，以及（ii）确定了两种最低限度水合中间体的结构，这些中间体被封闭在细胞外和细胞内环境中。
Neurotransmitter: sodium symporters (NSSs) regulate neuronal signal transmission by clearing excess neurotransmitters from the synapse, assisted by the co-transport of sodium ions. Extensive structural data have been collected in recent years for several members of the NSS family, which opened the way to structure-based studies for a mechanistic understanding of substrate transport. Leucine transporter (LeuT), a bacterial orthologue, has been broadly adopted as a prototype in these studies. This goal has been elusive, however, due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment. We provide here for the first time a comprehensive description of the molecular events leading to substrate/Na+ release to the postsynaptic cell, including the structure and dynamics of the N-terminal segment using a combination of molecular simulations. Substrate and Na+-release follows an influx of water molecules into the substrate/Na+-binding pocket accompanied by concerted rearrangements of transmembrane helices. A redistribution of salt bridges and cation-π interactions at the N-terminal segment prompts substrate release. Significantly, substrate release is followed by the closure of the intracellular gate and a global reconfiguration back to outward-facing state to resume the transport cycle. Two minimally hydrated intermediates, not structurally resolved to date, are identified: one, substrate-bound, stabilized during the passage from outward- to inward-facing state (holo-occluded), and another, substrate-free, along the reverse transition (apo-occluded). Bacterial leucine transporter (LeuT) belongs to neurotransmitter:sodium symporter (NSS) family. Its human orthologs include dopamine transporter and serotonin transporter. Malfunction of NSS members has been implicated in neurological diseases, hence the significance of elucidating their mechanism of function as clinically relevant drug targets. NSSs co-transport substrates (neurotransmitters or amino acids) and sodium ions across the cell membrane via alternating access to extracellular and intracellular media, which enables the uptake of substrate and ions from the extracellular region and their release to the intracellular region. Despite significant progress in elucidating the structure and function of NSS family members, their mechanism of function and the role of their N-terminal segment exposed to the cell interior remain elusive. Here, we provide for the first time a full-atomic time-resolved description of the complete transport cycle of LeuT using multiscale simulations. Two major findings are (i) elucidation of the structure and dynamics of the N-terminal segment which helps in mediating substrate and cation release and resuming the transport cycle, and (ii) determination of the structures of two minimally hydrated intermediates occluded to both extracellular and intracellular environments.
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影响因子：
3.3
作者：
Klauda, Jeffery B.;Venable, Richard M.;Freites, J. Alfredo;O'Connor, Joseph W.;Tobias, Douglas J.;Mondragon-Ramirez, Carlos;Vorobyov, Igor;MacKerell, Alexander D., Jr.;Pastor, Richard W.
通讯作者：
Pastor, Richard W.
影响因子：
64.8
作者：
Perez, Camilo;Koshy, Caroline;Ziegler, Christine
通讯作者：
Ziegler, Christine
影响因子：
5.8
作者：
Eyal, Eran;Yang, Lee-Wei;Bahar, Ivet
通讯作者：
Bahar, Ivet
影响因子：
4.3
作者：
Koldsø H;Noer P;Grouleff J;Autzen HE;Sinning S;Schiøtt B
通讯作者：
Schiøtt B
影响因子：
2.9
作者：
HOOVER, WG
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HOOVER, WG