Mechanisms of Biological Fluoride Resistance Exporters

描述(由申请人提供)：该项目将对两个新发现的F--转运膜蛋白家族进行详细的功能和机制研究，并将寻求解决它们的高分辨率晶体结构。这些蛋白质将F-从细胞质中排出，以保护细菌和单细胞真核生物免受环境中F-的毒性影响。这两个蛋白质家族在系统发育上没有亲缘关系。“Clcf”转运体是研究已久的CLC负离子通道和转运蛋白超家族中的一个分支，而“Fluc”转运体是一个功能未知的小分子膜蛋白家族。我们的初步实验已经为CLCF出口商发现了几个令人惊讶的变化，这些变化是在“传统的”CLC蛋白质中为氯-的运输确立的机制主题：(1)在所有以前研究的CLC中没有保守的阴离子配位残基，(2)对F-的极高的选择性，(3)尽管签名序列表明这些可能是离子通道，(3)质子耦合的F-反向端口机制，以及(4)前所未有的1：1阴离子/H+交换化学计量比。对于Fluc蛋白，我们的工作表明它们是高度选择F的离子通道。序列分析有力地证明，功能通道是一种不寻常的“反平行低聚物”，我们的实验结果表明，前所未有的二聚体结构中，孪生亚基以相反的方向插入膜中。该项目结合了电生理、膜生物物理和结构分析，以解决由这些结果引起的基本问题：什么残基决定了CLCF逆向转运体中的阴离子选择性和H+移动？我们必须如何修改公认的反迁移机制，以解释CLCFs令人惊讶的1：1的F-/H+化学计量比？熔剂通道中的孔衬残留物在哪里？是什么原因导致它们具有高的阴离子选择性？对这些基本问题的回答是必要的，以使我们了解这些膜蛋白是如何工作来输出氟的，从而抵消这种离子对细胞完整性的普遍挑战。由于这些F-出口因子存在于许多细菌和真核生物的病原体中，而不存在于脊椎动物中，因此它们可能提供新的抗生素靶点。

DESCRIPTION (provided by applicant): This project will subject two newly discovered families of F- -transporting membrane proteins to detailed functional and mechanistic scrutiny and will seek to solve their high-resolution crystal structures. These proteins expel F- from the cytoplasm to protect bacteria and unicellular eukaryotes from the toxic effects of ambient F- in the environment. The two protein families are phylogenetically unrelated. The "CLCF" exporters represent a clade within the long-studied CLC superfamily of anion channels and transporters, while the "Fluc" exporters are a previously unknown-function family of small membrane proteins. Our preliminary experiments have already uncovered for the CLCF exporters several surprising variations on mechanistic themes well-established for Cl- transport in "conventional" CLC proteins: (1) the absence of the anion- coordinating residues conserved among all previously studied CLCs, (2) an extremely high selectivity for F-, (3) a proton-coupled F- antiport mechanism despite a signature sequence suggesting that these would be ion channels, and (4) an unprecedented 1-to-1 anion/H+ exchange stoichiometry. For Fluc proteins, our work shows these to be highly F--selective ion channels. Sequence analysis argues strongly that the functional channel is an unusual "antiparallel oligomer," and our experimental results indicate unprecedented dimeric architecture in which the twin subunits are inserted into the membrane in opposite orientations. The project combines electrophysiological, membrane-biophysical, and structural analysis to attack fundamental questions arising from these results: what residues determine anion- selectivity and H+ movement in the CLCF antiporters? How must we modify accepted antiport mechanisms to account for the surprising 1-to-1 F-/H+ stoichiometry of CLCFs? Where are the pore-lining residues in Fluc channels and what accounts for their high anion selectivity? Answers to basic questions like these are necessary to bring into focus our view of how these membrane proteins work to export F- and thus counteract this ion's pervasive challenge to cellular integrity. Since these F- exporters are found in many bacterial and eukaryotic pathogens but not in vertebrates, they may provide novel antibiotic targets.