PDF(Pubmed)
Abstract：
Cell pH and Na+ homeostasis requires Na+/H+ antiporters. The crystal structure of NhaA, the main Escherichia coli Na+/H+ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III-V and X-XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li+ (the Na+ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.
摘要：
细胞pH和Na+稳态需要Na+/H+反转运蛋白。NhaA的晶体结构,主要的大肠杆菌Na+/H+反转运蛋白，揭示了原核和真核膜蛋白共有的独特的NhaA结构折叠。在12个NhaA跨膜节段(TMs)中，TMsIII-V和X-XII是拓扑反转的重复序列,其中展开的TMsIV和XI形成表征NhaA折叠的X形。我们表明，在NhaA突变体的氧化条件下，分子内交联在杂交中具有两个Cys置换（D133C-T340C）抑制了反转运蛋白活性，并损害了高盐中NhaA依赖性细胞的生长。亲和纯化的D133C-T340C蛋白在还原条件下结合Li+(NhaA的Na+替代底物)。交联将反载体捕获在面向外的构象中，阻止反端口循环。由于许多二级运输者被发现共享NhaA折叠，包括一些与人类疾病有关的，我们的数据对基础和临床研究都很重要.
