Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights.
Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights.
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DOI：
10.1085/jgp.201411219
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发表时间：
2014-12
期刊：
影响因子：
--
通讯作者：
Cameron AD
中科院分区：
文献类型：
--
作者：
Lee C;Yashiro S;Dotson DL;Uzdavinys P;Iwata S;Sansom MS;von Ballmoos C;Beckstein O;Drew D;Cameron AD
钠-质子反向转运蛋白NhaA的二聚体结构提供了Asp 163和Lys 300在转运机制中的作用的见解。钠-质子反向转运蛋白快速交换质子和钠离子穿过膜以调节细胞内pH、细胞体积和钠浓度。离子结合和释放如何与运输相关的构象变化相结合尚不清楚。在这里，我们报告的晶体形式的原型钠质子反向转运蛋白NhaA从大肠杆菌中的蛋白质被视为二聚体。在这个新的结构中，我们观察到一个必需的天冬氨酸（Asp 163）和保守的赖氨酸（Lys 300）之间的盐桥。一个等价的盐桥存在于同源转运蛋白NapA中，但不是唯一的其他已知的NhaA晶体结构中，这为大多数现有的产电钠-质子反向转运结构模型提供了基础。分子动力学模拟表明，盐桥的稳定性被削弱的钠离子结合到Asp 164和相邻的Asp 163。这表明转运机制涉及Asp 163在与Lys 300形成盐桥和与钠离子相互作用之间切换。pKa计算表明，Asp 163参与盐桥时极不可能被质子化。正如以前已经提出的，Asp 163是通过质子运输发生的两个残基之一，这些结果有明确的影响，目前的机制模型钠质子反向运输NhaA。
A dimeric structure of the sodium–proton antiporter NhaA provides insight into the roles of Asp163 and Lys300 in the transport mechanism. Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.
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