DeChancie Jason, Shrivastava Indira H, Bahar Ivet
Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, 3064 BST3, 3501 Fifth Ave, Pittsburgh, PA 15213, USA.
Mol Biosyst. 2011 Mar;7(3):832-42. doi: 10.1039/c0mb00175a. Epub 2010 Dec 15.
Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, Glt(Ph), has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of Glt(Ph). In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na(+) to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate-protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na(+), the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.
谷氨酸转运体通过将其底物（天冬氨酸或谷氨酸）转运至细胞内介质与钠离子或其他阳离子的能量有利转运相偶联，从而调节跨神经元和神经胶质细胞膜的兴奋性氨基酸神经传递。该家族首个通过晶体学解析的结构——古细菌天冬氨酸转运体Glt(Ph)，已成为阐明这些转运体底物转运机制的结构范例。构成该膜蛋白的三个亚基核心结构域中的两个螺旋发夹结构，即HP2和HP1，被认为分别作为底物摄取和释放的细胞外门和细胞内门。使用外向型结构进行的分子动力学模拟证实，HP2环作为细胞外门起作用。然而，由于缺乏结构数据，直到最近确定了Glt(Ph)的内向型结构，底物在原子尺度上的释放机制仍不清楚。在本研究中，我们利用这个最近解析的结构来模拟底物向细胞质的释放以及HP1和HP2在此过程中的作用。观察到高度灵活的HP2环作为激活剂（或启动剂），促使守门人Na(+)释放到细胞质中，并促进水分子从细胞质流入，这有效地破坏了底物与蛋白质的相互作用，并驱动底物从其结合位点上脱落。然而，底物释放和排出的完成还需要高度稳定的HP1环打开。总体而言，HP2环独特的构象灵活性、Na(+)的解离、结合口袋的水化以及HP1环3 - Ser基序的最终打开，成为控制谷氨酸转运体将结合底物释放到细胞内部的连续事件。
