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.

铜稳态失调会对人类健康产生多种后果。 哺乳动物细胞使用铜泵来维持稳态，金属化铜依赖性 酶，并将铜从一个细胞器/细胞/组织转移到另一个细胞器/细胞/组织具有同等的意义。 细菌转运蛋白及其作为病原体毒力因子的作用的研究非常重要。 存在于许多革兰氏阴性病原微生物中的一类外排泵是 RND- 型重金属输出者，以大肠杆菌的 CusCBAF 系统为例，该系统跨越 周质空间并表现出对 Cu(I) 和 Ag(I) 输出的选择性。 由三种蛋白质组成，CusA 是跨膜泵，CusB 是可溶性“适配器”蛋白质， CusC，一种外膜孔。此外，出口机械需要存在。 CusF，一种小的可溶性周质伴侣，可将 Cu 或 Ag 运送到三方复合物。 尽管近年来取得了许多进展，但这个难题的一个关键部分仍然缺失—— 金属转移的速率，以及该速率如何由特定的结构元素控制 PI 的实验室开发了一套动力学工具。 硒代蛋氨酸 (SeM) 取代与快速冷冻淬灭 (RFQ) 相结合，涉及混合和 该提案旨在应用这些工具进行研究。 CusCBAF 系统的金属输出机制在分子细节上有三种。 目标 1 将探索 CusF 的独特配位化学，其中包括 未加帽的色氨酸配体 (W44)，可加帽该位点并产生荧光 CO 与 W44A 变体结合的新发现将用于测试 490 nm 处的发射。 目标 2 将使用 W44 的作用是保护该位点免受 O2 氧化的假设。 RFQ 混合与 Se 边缘 Se-Cu/Ag 的 XAS 检测相结合，以跟踪金属动力学 从 CusF 转移到 CusB 以及从 CusF 转移到 CusA 以确定残留物 对于通过计算预测的转移复合物的效率很重要。 目标 3 将对蛋白质-蛋白质相互作用的研究进行分离和表征。 组氨酸激酶 CuS 周质结构域的金属传感机制 通过自磷酸化和磷酸转移激活 CusR 转录因子，测试 假设感觉域使用高亲和力和低亲和力金属结合位点来开启 并上调其激酶活性。预期结果是了解 分子细节中的金属输出机制，是制定策略的先决条件 用于中断致病金属抗性途径，降低毒力。