Mechanisms of Biological Fluoride Resistance Exporters

生物耐氟输出体的机制

• 批准号: 8680494
• 负责人: Christopher Miller
• 金额: $ 28.18万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8680494
• 关键词: Accounting; Affinity; Amino Acid Sequence; Anions; Antibiotics; Architecture; Bacteria; Basic Science; Bathing; Behavior; Biological; Biological Process; CLC Gene; CLC-1 channel; Candida albicans; Carrier Proteins; Categories; Chemistry; Cone; Coupled; Coupling; Crystallization; Cytoplasm; Databases; Employee Strikes; Enterococcus; Environment; Enzymes; Eukaryota; Exhibits; Fab Immunoglobulins; Family; Fibronectins; Fluoride Ion; Fluorides; Genes; Genome; Glycolysis; Homologous Gene; Investigation; Ion Channel; Ion Transport; Ions; Leishmania major; Light; Membrane; Membrane Proteins; Membrane Transport Proteins; Molecular; Molecular Chaperones; Movement; Mycobacterium tuberculosis; Nucleic Acids; Oral health; Organism; Peptide Sequence Determination; Property; Protein Family; Proteins; Protons; Protozoa; Research; Resistance; Resolution; Sea; Sequence Analysis; Soil; Specificity; Staging; Structure; Surveys; Testing; Time; Toxic effect; Toxin; Toxoplasma gondii; Twin Multiple Birth; Variant; Vertebrates; Water fluoridation; Work; Xenobiotics; antiport; antiporter; base; drinking water; duplicate genes; falls; fungus; improved; insight; member; microorganism; novel; pathogen; public health relevance; reconstitution; research study; stoichiometry; vertebrate genome

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.

项目概要 该项目将对两个新发现的 F- 转运膜蛋白家族进行研究 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 系统发育无关。 “CLCF”出口商代表了长期研究的 CLC 中的一个分支 阴离子通道和转运蛋白的超家族，而“Fluc”输出蛋白是以前的 功能未知的小膜蛋白家族。我们的初步实验有 已经为 CLCF 出口商发现了机械主题的几个令人惊讶的变化 “传统”CLC 蛋白中的 Cl- 转运已得到充分证实：(1) 不存在阴离子 在所有先前研究的 CLC 中保守的配位残基，(2) 极高 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 表明这些将是离子通道，并且 (4) 前所未有的 1 对 1 阴离子/H+ 交换化学计量。对于 Fluc 蛋白，我们的工作表明它们具有高度 F-选择性离子 渠道。序列分析有力地证明了功能通道是一个不寻常的通道 “反平行寡聚体”，我们的实验结果表明前所未有的二聚体 双亚基以相反方向插入膜中的结构。 该项目结合了电生理学、膜生物物理和结构分析 解决这些结果产生的基本问题：什么残留物决定阴离子 CLCF 反向转运蛋白的选择性和 H+ 运动？我们必须如何修改已接受的反端口 解释 CLCF 令人惊讶的 1:1 F-/H+ 化学计量的机制是什么？哪里有 Fluc 通道中的孔衬残留物及其高阴离子选择性的原因是什么？ 回答诸如此类的基本问题对于让我们聚焦于这些问题的看法是必要的。 膜蛋白致力于输出 F-，从而抵消该离子对 细胞完整性。由于这些 F- 输出蛋白存在于许多细菌和真核生物中 病原体但不在脊椎动物中，它们可能提供新的抗生素靶标。