The pH sensor and ion binding of NhaD Na + /H + antiporter from IT superfamily
The pH sensor and ion binding of NhaD Na + /H + antiporter from IT superfamily
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IT 超家族 NhaD Na /H 反向转运蛋白的 pH 传感器和离子结合
DOI：
10.1111/mmi.14965
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发表时间：
2022
影响因子：
3.6
通讯作者：
Chunyu Yang
中科院分区：
文献类型：
--
作者：
Peiwen Lv;Yaru Li;Ruimin Wang;Ye Zhang;Wenkai Wang;Yuanxiang Liu;Yan Shang;D;an Su;Wei Wang;Chunyu Yang
离子转运蛋白(IT)超家族的钠-质子(Na+/H+)逆向转运蛋白在控制pH和电解质动态平衡方面起着至关重要的作用。然而，到目前为止，关于它们的结构功能的信息非常有限。在本研究中，NHAD反向转运体的结构模型被认为是IT蛋白的典型发夹结构，核心底物结合位点上有两个对称保守的支架结构域，并确定了四个基序。此外，涉及这些结构域的25个保守位点受到定点突变，所有突变都导致运输能力的影响。特别是，作为Na+结合位点的候选者，发夹不连续的D166和D405突变不利于转运活性，并通过荧光共振能量转移(FRET)分析发现引起显著的构象变化。此外，如在NhaA结构中观察到的，一些带电残基，如E64、E65、R454和R464，被预测参与NHAD激活的净电荷开关，在细胞质漏斗的入口处共同形成“pH传感器”。围绕这些残基的突变不利于NHAD的转运活性，或失去对pH信号的响应能力，并引发Na+转运的构象变化。
Sodium‐proton (Na+/H+) antiporters from the ion transporter (IT) superfamily play a vital role in controlling the pH and electrolyte homeostasis. However, very limited information regarding their structural functions is available to date. In this study, the structural model of the NhaD antiporter was proposed as a typical hairpin structure of IT proteins, with two symmetrically conserved scaffold domains that frame the core substrate‐binding sites, and four motifs were identified. Furthermore, 25 conserved sites involving these domains were subjected to site‐directed mutagenesis, and all mutations resulted in an impact on transport abilities. In particular, as candidates for Na+‐binding sites, D166 and D405 mutations at hairpin discontinuities were detrimental to transport activities and were found to induce pronounced conformational changes using fluorescence resonance energy transfer (FRET) assays. In addition, as observed in the NhaA structure, some charged residues, for example, E64, E65, R454, and R464, are predicted to be involved in the net charge switch of NhaD activation, by collectively form a “pH sensor” at the entrance of the cytoplasmic funnel. Mutations encompassing these residues were detrimental to the transport activity of NhaD or lost the capacity to respond to pH signals and trigger conformational changes for Na+translocation.