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Structure and mechanism of the FocA formate channel: Discovery of an organic ion channel.


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Transport of charged and polar solutes across membranes is fundamental to cellular organisms, and studies on these processes are elemental to the understanding of a wide variety of biological fields ranging from cellular metabolism to neurology. Integral membrane channel proteins represent basic systems of solute transport, catalyzing the translocation of their substrates down a concentration gradient without the need for external energy sources. Nevertheless, channel proteins exhibit a high degree of substrate selectivity and yet can facilitate ionic flux rates that often approach the limits of diffusion.;In anaerobic environments, microorganisms convert up to one third of metabolized glucose into the simple carboxylate ion, formate. In the Escherichia coli bacterium, this metabolite is transported across the inner membrane through the FocA protein, which serves as the founding member in the formate/nitrite transporter (FNT) family encompassing over 1500 homologues in bacteria, yeast and simple eukaryotes. Additionally, a nitrite transporting variant of the FNT family has been shown in mice to play a role in Salmonella pathogenesis and may provide a paradigm for bacterial defense against reactive nitrogen species released by the host.;High resolution structural data for any FNT family members has been unavailable, and the mechanism of substrate translocation has not been determined. In this work, the successful purification, crystallization and subsequent structure elucidation of the novel FocA protein from Vibrio cholerae structure to 2.1 A resolution is described. A supplemental crystal structure shows the location of two substrate molecules near a central and conserved region of the cytoplasmic pore, and biochemical assays support activity of the protein.;The tertiary protein structure exhibits a homopentameric organization, however the monomeric topology and fold is represented in the tetrameric aquaporin family of water and glycerol channels. Within the crystallographic pentamer, a conserved half-membrane spanning pore helix adopts different conformations. In addition, the location of substrate molecules in this region indicates that the FocA selectivity filter is formed on the cytoplasmic side of the protein and that lateral gating of the pore region is activated by the presence of formate molecules. A hypothesis for selectivity and gating in the FNT family is presented.
机译:带电和极性溶质跨膜的运输是细胞生物的基础,对这些过程的研究对于理解从细胞代谢到神经病学等众多生物学领域都是至关重要的。完整的膜通道蛋白代表溶质运输的基本系统,可在不需要外部能源的情况下催化其底物沿浓度梯度的转运。尽管如此,通道蛋白仍具有很高的底物选择性,并且可以促进离子通量速率,而离子通量速率通常接近扩散极限。在厌氧环境中,微生物最多将三分之一的代谢葡萄糖转化为简单的羧酸根离子甲酸盐。在大肠杆菌中,这种代谢物通过FocA蛋白跨内膜运输,而FocA蛋白是甲酸/亚硝酸盐转运蛋白(FNT)家族的创始成员,涵盖细菌,酵母和简单的真核生物中的1500多个同源物。此外,已显示FNT家族的亚硝酸盐转运变体在小鼠沙门氏菌的发病机理中发挥作用,并可能为细菌防御宿主释放的活性氮提供了范例;任何FNT家族成员的高分辨率结构数据都有尚不可用,并且底物转运的机制尚未确定。在这项工作中,描述了从霍乱弧菌结构到2.1 A分辨率的新型FocA蛋白的成功纯化,结晶和随后的结构阐明。补充的晶体结构显示了两个底物分子在细胞质孔的中央和保守区域附近的位置,并且生化分析支持了该蛋白的活性。三级蛋白结构表现出同五聚体结构,但是单体拓扑结构和折叠表示为水和甘油通道的四聚体水通道蛋白家族。在晶体五聚体中,跨孔螺旋的保守半膜采用不同的构象。此外,底物分子在该区域中的位置表明FocA选择性滤膜形成在蛋白质的细胞质侧,并且通过甲酸分子的存在激活了孔区域的横向门控。提出了FNT家族中选择性和门控的假设。


  • 作者

    Waight, Andrew Bryan.;

  • 作者单位

    New York University.;

  • 授予单位 New York University.;
  • 学科 Biology Cell.;Chemistry Biochemistry.;Biophysics Medical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 241 p.
  • 总页数 241
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:36:56


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