Metal reservoirs & intracellular fluxes : characterization of three chaperone proteins contributing to zinc homeostasis in Cupriavidus metallidurans

金属储液罐

• 批准号: 328566663
• 负责人: Professor Dr. Dietrich H. Nies
• 金额: --
• 依托单位: Abteilung Pharmazeutische Chemie & Bioanalytik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/328566663?language=en
• 关键词: Metal; reservoirs; intracellular; fluxes; characterization

The beta-proteobacterium Cupriavidus metallidurans strain CH34 is able to survive in the presence of high concentration of individual heavy metals or a mixture of them, such as Zn(II), Co(II), Ni(II), Cd(II), Cu(II), Ag(I) and Au(III) complexes. C. metallidurans is present in various metal-contaminated environments around the world, e.g. metal-rich auriferous soils in Australia or serpentine soils in New Caledonia. It is therefore a model organism to study how cells are able to use transition metal cations for their cellular biochemistry, despite their high toxicity. During the last decades, we have revealed how the metal transportome of Cupriavidus metallidurans, the totality of its metal uptake and efflux systems, transforms the metal content and speciation in the environment into the cytoplasmic metal mélange. This is achieved by a combination of a rather unspecific import of a broad spectrum of metal cations in combination with a specific removal of surplus cations by efflux systems, some exporting their substrates from the periplasm and some from the cytoplasm. When transition metal import was studied more closely, it could be demonstrated that the actual import route influences the efficiency of metal allocation, e.g. of zinc to the important RpoC (beta-prime) subunit of the bacterial RNA polymerase. This indicated the presence of specific metal delivery channels and metal pools in C. metallidurans but the big picture of the interplay between uptake, efflux, storage and specific allocation mechanisms is still not framed. The role in metal homeostasis of three potentially zinc-binding metal chaperones and GTPases (CobW1 to CobW3) from C. metallidurans will be characterized here to understand the allocation of zinc ions and the contribution of the three CobW proteins to this process. CobW1 is needed to supply zinc under zinc starvation conditions, CobW2 may be a zinc storage protein and part of the zinc repository, while CobW3 is no GTPase but may interact with zinc import systems. A bottom-up approach will lead from biochemical and structure studies to interactome experiments done by cross-linked mass spectrometry (XL-MS) in cooperation with Andrea Sinz, Uni Halle. This bottom-up approach will start with a detailed biochemical characterization of the three CobW proteins by isothermal calorimetry, XL-MS of important conformations leading to crystallization attempts or NMR-studies of the most important protein conformations (year 1). In the second year, interaction with selected partner proteins will be studied by metal delivery assays, biacore experiments and XL-MS. In the third year, the interactome of selected C. metallidurans strains cultivated under different conditions will be studied by XL-MS. This bottom-up approach will be paralleled by a top-down approach using cell fractionation and pulse-chase experiments to determine the biochemical nature of the zinc pools in C. metallidurans.

β-变形菌 Cupriavidus metallidurans 菌株 CH34 能够在高浓度的单一重金属或它们的混合物存在下生存，例如 Zn(II)、Co(II)、Ni(II)、Cd(II)、Cu(II)、Ag(I) 和 Au(III) 复合物。 C. Metallidurans 存在于世界各地各种金属污染的环境中，例如：澳大利亚富含金属的含金土壤或新喀里多尼亚的蛇纹石土壤。因此，它是研究细胞如何利用过渡金属阳离子进行细胞生物化学的模型生物，尽管它们具有高毒性。在过去的几十年中，我们揭示了Cupriavidus metallidurans的金属转运组，即其金属摄取和流出系统的整体，如何将环境中的金属含量和形态转化为细胞质金属混合物。这是通过将相当非特异性的广谱金属阳离子导入与通过外排系统特异性去除多余阳离子相结合来实现的，其中一些从周质输出其底物，一些从细胞质输出。当对过渡金属进口进行更仔细的研究时，可以证明实际的进口路线会影响金属配置的效率，例如：锌与细菌 RNA 聚合酶重要的 RpoC（β-prime）亚基的结合。这表明 C. Metallidurans 中存在特定的金属输送通道和金属池，但摄取、流出、储存和特定分配机制之间相互作用的总体情况仍未确定。此处将表征来自 C. metallidurans 的三种潜在锌结合金属伴侣和 GTPases（CobW1 至 CobW3）在金属稳态中的作用，以了解锌离子的分配以及三种 CobW 蛋白对此过程的贡献。在缺锌条件下，需要 CobW1 来供应锌，CobW2 可能是锌储存蛋白，也是锌库的一部分，而 CobW3 不是 GTP 酶，但可能与锌输入系统相互作用。自下而上的方法将从生化和结构研究到与哈勒大学的 Andrea Sinz 合作通过交联质谱 (XL-MS) 进行的相互作用组实验。这种自下而上的方法将从通过等温量热法对三种 CobW 蛋白进行详细的生化表征开始，重要构象的 XL-MS 导致对最重要的蛋白质构象进行结晶尝试或 NMR 研究（第一年）。第二年，将通过金属递送测定、biacore 实验和 XL-MS 研究与选定伙伴蛋白的相互作用。第三年，将通过 XL-MS 研究在不同条件下培养的选定 C. metallidurans 菌株的相互作用组。这种自下而上的方法将与自上而下的方法并行，使用细胞分级分离和脉冲追踪实验来确定 C. metallidurans 中锌池的生化性质。