Mechanisms of Biological Fluoride Resistance Exporters

生物耐氟输出体的机制

• 批准号: 8891459
• 负责人: Christopher Miller
• 金额: $ 28.25万
• 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/8891459
• 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; Health; 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; reconstitution; research study; stoichiometry; vertebrate genome

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.

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审查，并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- 的毒性作用。这两个蛋白质家族在系统发育上是无关的。 “CLCF”输出蛋白代表了长期研究的阴离子通道和转运蛋白 CLC 超家族中的一个分支，而“Fluc”输出蛋白是一个先前未知功能的小膜蛋白家族。我们的初步实验已经为 CLCF 输出者揭示了“传统”CLC 蛋白中 Cl-转运的机械主题的几个令人惊讶的变化：(1) 在所有先前研究的 CLC 中不存在保守的阴离子配位残基，(2 ）对F-具有极高的选择性，（3）质子耦合的F-反端口机制，尽管特征序列表明这些将是离子通道，以及（4）前所未有的1:1 阴离子/H+ 交换化学计量。对于 Fluc 蛋白，我们的工作表明它们是高度 F-选择性离子通道。序列分析强烈认为功能通道是一种不寻常的“反平行寡聚物”，我们的实验结果表明前所未有的二聚体结构，其中双亚基以相反的方向插入膜中。该项目结合了电生理学、膜生物物理和结构分析来解决这些结果产生的基本问题：什么残基决定 CLCF 反向转运蛋白中的阴离子选择性和 H+ 运动？我们必须如何修改公认的反端口机制来解释 CLCF 令人惊讶的 1:1 F-/H+ 化学计量？ Fluc 通道中的孔衬残留物在哪里？是什么导致了它们的高阴离子选择性？对这些基本问题的回答对于我们了解这些膜蛋白如何输出 F- 从而抵消该离子对细胞完整性的普遍挑战至关重要。由于这些 F- 输出蛋白存在于许多细菌和真核病原体中，但在脊椎动物中没有，因此它们可能提供新的抗生素靶点。