The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly alpha-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.

中文翻译：

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一株希瓦氏菌抗砷家庭转运蛋白的结构和功能研究。

有毒的准金属砷是一种丰富的元素，大多数生物都具有涉及其排毒的转运系统。一种这样的亚砷转运蛋白家族，即ACR3家族，广泛存在于真菌和细菌中。为了更好地了解砷转运的分子机制，我们在这里报告了来自沙伊氏菌oneidensis MR-1的家族成员So_ACR3的表达和特征。出人意料的是，这种转运蛋白在砷敏感性高的大肠杆菌菌株AW3110中的表达赋予了对砷酸盐的抗性，但对砷酸盐没有抗性。纯化蛋白的C末端带有His标签的形式可以直接测试假定的渗透物的结合：砷酸盐而不是砷酸盐以浓度依赖的方式猝灭了其固有的荧光。傅立叶变换红外光谱表明，纯化的蛋白质主要是α-螺旋。在第3位带有单个半胱氨酸残基的突变体保留赋予砷抗性的能力，并且在完整细胞中膜不渗透硫醇试剂可以接近。结合成功的低聚组氨酸C末端标记，这一发现与实验确定的同源人类根尖钠依赖性胆汁酸转运蛋白的拓扑结构一致，即7个跨膜螺旋和一个周质N末端，尽管存在其他跨膜细分不能排除。在第四个推定的跨膜区域中，保守残基脯氨酸190向丙氨酸的突变消除了转运蛋白赋予砷抗性的能力，但并未阻止砷酸盐的结合。