Time-resolved mechanism of extracellular gate opening and substrate binding in a glutamate transporter.
Time-resolved mechanism of extracellular gate opening and substrate binding in a glutamate transporter.
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DOI：
10.1074/jbc.m800889200
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发表时间：
2008-10-17
期刊：
影响因子：
--
通讯作者：
Bahar I
中科院分区：
文献类型：
--
作者：
Shrivastava IH;Jiang J;Amara SG;Bahar I
谷氨酸转运蛋白，也被称为兴奋性氨基酸转运蛋白（EAATs），是一种调节谷氨酸能信号传递的膜蛋白，在突触释放过量的谷氨酸后清除谷氨酸。直到最近确定了一种古细菌转运体GltPh的x射线结构，对其分子功能机制的基于结构的理解才变得难以捉摸。GltPh以三聚体形式存在，每个亚基包含一个介导底物易位的核心区域。本研究根据新的实验数据对eaat的底物结合特性进行了一系列的分子动力学模拟和分析。模拟首次提供了驱动底物识别和结合的时间分辨事件的完整原子描述。每个亚基的核心区域表现出内在的倾向，在几十纳秒内打开螺旋发夹HP2环，即细胞外门，暴露保守的极性残基，作为底物结合的吸引子。跨膜螺旋TM7部分未缠绕部分的NMDGT基序和TM8上的Asp-390和Asp-394残基也因其在底物结合和与介导水分子和/或钠离子的密切相互作用中发挥重要作用而被区分。模拟结果显示，在TM7上的Leu-303和TM8上的Asp-405中有一个Na+结合位点，并支持钠离子在稳定底物结合构象中的作用。通过对hEAAT1突变体L391C和L391A进行定点诱变和Na+依赖性试验，证实了Leu-303或其对应物Leu-391在人EAAT1 （hEAAT1）中的功能重要性。
Glutamate transporters, also referred to as excitatory amino acid transporters (EAATs), are membrane proteins that regulate glutamatergic signal transmission by clearing excess glutamate after its release at synapses. A structure-based understanding of their molecular mechanisms of function has been elusive until the recent determination of the x-ray structure of an archaeal transporter, GltPh. GltPh exists as a trimer, with each subunit containing a core region that mediates substrate translocation. In the present study a series of molecular dynamics simulations have been conducted and analyzed in light of new experimental data on substrate binding properties of EAATs. The simulations provide for the first time a full atomic description of the time-resolved events that drive the recognition and binding of substrate. The core region of each subunit exhibits an intrinsic tendency to open the helical hairpin HP2 loop, the extracellular gate, within tens of nanoseconds exposing conserved polar residues that serve as attractors for substrate binding. The NMDGT motif on the partially unwound part of the transmembrane helix TM7 and the residues Asp-390 and Asp-394 on TM8 are also distinguished by their important role in substrate binding and close interaction with mediating water molecules and/or sodium ions. The simulations reveal a Na+ binding site comprised in part of Leu-303 on TM7 and Asp-405 on TM8 and support a role for sodium ions in stabilizing substrate-bound conformers. The functional importance of Leu-303 or its counterpart Leu-391 in human EAAT1 (hEAAT1) is confirmed by site-directed mutagenesis and Na+ dependence assays conducted with hEAAT1 mutants L391C and L391A.