Mechanisms of Biological Fluoride Resistance Exporters

项目总结 该项目将使两个新发现的F-转运膜蛋白家族 详细的功能和机械审查，并将寻求解决他们的高分辨率晶体 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。这两个蛋白质家族是 在系统发育上没有亲缘关系。“CLCF”出口商代表了长期研究的CLLC中的一个分支 阴离子通道和转运蛋白的超家族，而“Fluc”出口商是以前的 功能未知的小分子膜蛋白家族。我们的初步实验已经 已经为CLCF出口商发现了几个令人惊讶的机械论主题变化 氯离子在“常规”CLC蛋白中的转运：(1)阴离子的缺失。 配位残基在所有先前研究的CLC中保守，(2)极高的 F-的选择性，(3)尽管有签名序列，但质子耦合的F-反端口机制 这表明这些可能是离子通道，以及(4)前所未有的1：1的阴离子/H+ 交换化学计量学。对于Fluc蛋白，我们的工作表明它们是高度F-选择性的离子 频道。序列分析有力地论证了功能通道是一种不寻常的 “反平行齐聚物”，我们的实验结果表明，前所未有的二聚体 双胞胎亚单位以相反的方向插入细胞膜的结构。 该项目结合了电生理、膜生物物理和结构分析，以 攻击这些结果产生的基本问题：什么残基决定阴离子- CLCF逆向转运蛋白中的选择性和H+运动？我们必须如何修改已接受的反端口 解释CLCFs令人惊讶的1：1 F-/H+化学计量比的机制？在哪里？ 熔剂通道中的孔衬残留物以及它们高阴离子选择性的原因是什么？ 对这些基本问题的回答是必要的，以使我们了解这些问题是如何 膜蛋白的作用是输出F-，从而抵消这种离子对 细胞完整性。由于这些F-输出体存在于许多细菌和真核生物中 病原体，而不是在脊椎动物中，它们可能提供新的抗生素靶点。

Project summary 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.