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重影响。 哺乳动物细胞使用铜泵维持稳态，依赖金属铜 酶，然后将铜从一个细胞器/细胞/组织转移到另一种细胞器/细胞/组织。同等意义的是 细菌转运蛋白的研究及其在病原体中作为病毒因素的作用。一个重要的 在许多革兰氏阴性致病生物中存在的排出泵类是RND- 类型的重金属出口商由大肠杆菌的Cuscbaf系统举例说明 外围简单空间并具有出口Cu（i）和Ag（i）的选择性。该综合体是 CUSA跨膜泵完成了三种蛋白质，CUSB是一种可溶的“适配器”蛋白， 和CUSC，外膜孔。此外，出口机械需要存在 CUSF，一种小的可溶性周质伴侣，将Cu或Ag穿向三方复合物。 尽管近年来取得了许多进步，但困扰着一个重要的难题 - 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 涉及硒蛋氨酸（SEM）替代与快速冷冻淬火（RFQ）混合和 广泛适用的XAS检测。该提案旨在应用这些工具来学习 Cuscbaf系统的金属出口机理，分子细节。有三个 具体目标。 AIM 1将探索CUSF独特的协调化学，其中包括 前所未有的色氨酸配体（W44），该配体限制该位置并引起荧光 在490 nm处排放。 CO与W44A变体结合的新颖发现将用于测试 W44的作用是保护位点免受O2的氧化的假设。 AIM 2将使用 RFQ混合与XAS在SE边缘检测Se-Cu/ag的耦合以遵循金属的动力学 从CUSF转移到CUSB，从CUSF转移到CUSA，目的是确定保留率 对于金属出口的效率很重要。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将调查 组氨酸激酶cuss的周质结构域的金属传感机理 通过自噬和phophotransfer激活CUSR转录因子，测试 感觉域使用高亲和力金属结合位点开启的假设 并上调其激酶活性。预期的结果是对 金属出口机制以分子细节为单位，这是制定策略的先决条件 用于中断致病金属电阻途径，并减少病毒。