Revealing the Ligand Binding Site of NhaA Na+/H+ Antiporter and Its pH Dependence
Revealing the Ligand Binding Site of NhaA Na+/H+ Antiporter and Its pH Dependence
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DOI：
10.1074/jbc.m112.391128
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发表时间：
2012-11-02
影响因子：
4.8
通讯作者：
Padan, Etana
中科院分区：
文献类型：
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作者：
Maes, Michal;Rimon, Abraham;Padan, Etana
所有细胞的pH和Na+稳态都需要Na+/H+反向转运蛋白。在大多数情况下，它们的活性受到pH值的严格调节。NhaA是大肠杆菌的主要反向转运蛋白，在所有生物界中都有同源物。NhaA的晶体结构提供了对反向转运蛋白的作用机制和pH调节的见解。然而，NhaA的活性位点仍然难以捉摸，因为既没有Na+也没有Li+，NhaA配体，在结构中观察到。使用等温滴定量热法，我们表明，纯化的NhaA绑定Li+在洗涤剂胶束。这种相互作用是由焓的增加驱动的（在283 K下，Δ H为-8000 +/- 300 cal/mol，Δ S为-15.2 cal/mol/degree），涉及每个NhaA分子的单个结合位点，并且具有高度特异性，并且显著依赖于pH;仅在pH 8.5下观察到Li+结合。结合突变分析与等温滴定量热法测量显示，Asp-163，Asp-164，Thr-132和Asp-133形成Li+结合位点，而Lys-300在反向转运蛋白的pH调节中起重要作用。
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na+ nor Li+, the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li+ in detergent micelles. This interaction is driven by an increase in enthalpy (Delta H of -8000 +/- 300 cal/mol and Delta S of -15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li+ binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li+ binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.