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

铜绿假单胞菌CH34菌株能在单一或多种重金属的混合存在下存活，如锌(II)、钴(II)、镍(II)、镉(II)、铜(II)、银(I)和金(III)的络合物。金黄色葡萄球菌存在于世界各地各种金属污染的环境中，例如澳大利亚的富含金属的含金土壤或新喀里多尼亚的蛇纹石土壤。因此，它是研究细胞如何能够利用过渡金属阳离子进行细胞生化的模式生物，尽管它们具有很高的毒性。在过去的几十年里，我们揭示了金属铜绿假单胞菌的金属转运体，即其金属摄取和外排系统的整体，是如何将环境中的金属含量和形态转化为细胞质中的金属混合的。这是通过相当不特定的广谱金属阳离子的进口和外排系统对多余阳离子的特定去除相结合的方式实现的，其中一些从周质中输出底物，一些从细胞质中输出。当更仔细地研究过渡金属进口时，可以证明实际的进口路线会影响金属分配的效率，例如锌到细菌RNA聚合酶的重要RpoC(β-质数)亚单位的分配。这表明在金黄色葡萄球菌中存在特定的金属输送通道和金属库，但摄取、外排、储存和特定分配机制之间的相互作用的大图景仍未被框定。为了了解锌离子的分配和三种CobW蛋白在这一过程中的作用，我们将研究三种可能与锌结合的金属伴侣蛋白和GTP酶(CobW1到CobW3)在金属动态平衡中的作用。在缺锌条件下，CobW1是锌的供给者，CobW2可能是锌储存蛋白和锌储存库的一部分，而CobW3不是GTP酶，但可能与锌输入系统相互作用。自下而上的方法将导致从生化和结构研究到由交叉连接质谱仪(XL-MS)与Uni Halle的Andrea Sinz合作进行的交互作用组实验。这种自下而上的方法将首先通过等温量热法、导致结晶尝试的重要构象的XL-MS或最重要蛋白质构象的核磁共振研究(第1年)对三种CobW蛋白质进行详细的生化表征。在第二年，将通过金属传递分析、Biacore实验和XL-MS来研究与选定的伙伴蛋白的相互作用。在第三年，我们将用XL-MS研究在不同条件下培养的金黄色葡萄球菌菌株的相互作用组。这种自下而上的方法将与自上而下的方法并行，使用细胞分离和脉冲追逐实验来确定金属假单胞菌锌库的生化性质。

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.