Tracking Metal Flux Through a Pathogenic Export Complex

通过致病性出口复合物追踪金属通量

• 批准号: 9331067
• 负责人: Ninian J Blackburn
• 金额: $ 36.57万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9331067
• 关键词: Acute; Adaptor Signaling Protein; Aerobic; Affinity; Binding; Brain; Cells; Chemistry; Complex; Computer Simulation; Copper; Coupled; Detection; Development; Disease; Elements; Ensure; Enzymes; Escherichia coli; Exhibits; Fluorescence; Freezing; Goals; Gram-Negative Bacteria; Health; Heavy Metals; Hepatolenticular Degeneration; Homeostasis; Human; Immune response; Interruption; Invaded; Ions; Kinetics; Label; Laboratories; Lead; Ligands; Ligation; Liver; Mammalian Cell; Mediator of activation protein; Membrane; Menkes Kinky Hair Syndrome; Metal Binding Site; Metals; Methionine; Molecular; Molecular Chaperones; Mutation; Organelles; Organism; Outcome; Oxidation-Reduction; Oxygen; Pathogenicity; Pathway interactions; Phagolysosome; Phosphotransferases; Play; Process; Proteins; Pump; Regulation; Research; Resistance; Role; Selenomethionine; Sensory; Set protein; Site; Spectroscopy, Fourier Transform Infrared; Syndrome; System; Testing; Tissues; Tryptophan; Up-Regulation; Variant; Virulence; Virulence Factors; Wilson disease protein; antimicrobial drug; computer studies; efflux pump; hypocupremia; innovation; killings; macrophage; microbial; novel; oxidation; pathogen; periplasm; protein protein interaction; protein-histidine kinase; response; scaffold; sensor; tool; trafficking; transcription factor

Dysregulation of copper homeostasis has multiple consequences for human health. Mammalian cells use copper pumps to maintain homeostasis, metalate copper-dependent enzymes, and transfer copper from one organelle/cell/tissue to another. Of equal significance is the study of bacterial transporters and their role as virulence factors in pathogens. An important class of efflux pump, present in a number of gram-negative pathogenic organisms is the RND- type heavy metal exporter exemplified by the CusCBAF system of E. coli which spans the periplasmic space and exhibits selectivity for export of Cu(I) and Ag(I). The complex is comprised of three proteins, CusA a transmembrane pump, CusB is a soluble “adaptor” protein, and CusC, an outer-membrane pore. In addition, the export machinery requires the presence of CusF, a small soluble periplasmic chaperone which shuttles Cu or Ag to the tripartite complex. Despite many advances in recent years, a critical piece of the puzzle has been missing – the rate at which metals transfer, and how that rate is controlled by specific structural elements of the interacting protein components. The PI’s laboratory has developed a suite of kinetic tools involving selenomethionine (SeM) substitution coupled to rapid freeze quench (RFQ) mixing and XAS detection which are broadly applicable. This proposal sets out to apply these tools to study the mechanisms of metal export by the CusCBAF system in molecular detail. There are three specific aims. Aim 1 will explore the unique coordination chemistry of CusF, which includes an unprecedented tryptophan ligand (W44) that caps the site and gives rise to fluorescence emission at 490 nm. The novel finding that CO binds to the W44A variant will be used to test the hypothesis that the role of W44 is to protect the site from oxidation via O2. Aim 2 will use RFQ mixing coupled to XAS detection of Se-Cu/Ag at the Se edge to follow the kinetics of metal transfer from CusF to CusB and from CusF to CusA with the goal of determining residues important for the efficiency of metal export. Transfer complexes predicted from computational studies of protein-protein interactions will be isolated and characterized. Aim 3 will investigate the mechanism of metal sensing by the periplasmic domain of the histidine kinase CusS which activates the CusR transcription factor via autophosphorylation and phosphotransfer, testing the hypothesis that the sensory domain uses high and low-affinity metal binding sites to switch on and upregulate its kinase activity. The expected outcome is an understanding of the mechanism of metal export in molecular detail, a prerequisite for the development of strategies for interrupting pathogenic metal resistance pathways, and diminishing virulence.

铜稳态失调对人类健康有多种后果。 哺乳动物细胞使用铜泵来维持体内平衡，金属化铜依赖 酶，并将铜从一个细胞器/细胞/组织转移到另一个。同样重要的是 研究细菌转运体及其作为病原体中毒力因子的作用的学科。一个重要 存在于许多革兰氏阴性病原生物体中的外排泵类别是RND- 以E.大肠杆菌， 周质空间，并表现出选择性输出的Cu（I）和Ag（I）。述复合物 由三种蛋白质组成，CusA是跨膜泵，CusB是可溶性“接头”蛋白， 和外膜孔CusC。此外，出口机制还需要 CusF是一种小分子可溶性周质分子伴侣，可将Cu或Ag转运至三联复合体。 尽管近年来取得了许多进展，但仍缺少一个关键的难题-- 金属转移的速率，以及该速率如何由特定的结构元素控制， 相互作用的蛋白质成分。PI的实验室开发了一套动力学工具 涉及硒代甲硫氨酸（SeM）取代与快速冷冻淬灭（FHT）混合， XAS检测具有广泛的适用性。本提案旨在将这些工具应用于研究 CusCBAF系统的金属输出机制的分子细节。有三 明确的目标。目的1将探索CuSF的独特配位化学，其中包括 一种前所未有的色氨酸配体（W 44），覆盖该位点并产生荧光 发射波长490 nm。CO与W 44 A变体结合的新发现将用于测试 假设W 44的作用是通过O2保护位点不被氧化。目标2将使用 在Se边缘处的Se-Cu/Ag的混合混合耦合到XAS检测以跟踪金属的动力学 从CusF转移到CusB和从CusF转移到CusA，目的是测定残留物 这对金属出口的效率至关重要。从计算中预测的转移复合物 蛋白质-蛋白质相互作用的研究将被分离和表征。Aim 3将进行调查 组氨酸激酶CusS的周质结构域的金属传感机制， 通过自身磷酸化和磷酸转移激活CusR转录因子，测试 假设感觉域使用高和低亲和力金属结合位点来开启 并上调其激酶活性。预期的结果是了解 金属出口机制的分子细节，为战略的发展的先决条件 用于中断致病性金属抗性途径和降低毒性。