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

Cupriavidus metallidurans菌株CH34能够在高浓度的单个重金属或它们的混合物中生存，例如Zn(II), Co(II), Ni(II), Cd(II), Cu(II), Ag(I)和Au（III）配合物。C. metallidurans存在于世界各地各种金属污染的环境中，例如澳大利亚的富金属含金土壤或新喀里多尼亚的蛇纹石土壤。因此，它是一种模式生物，研究细胞如何能够利用过渡金属阳离子进行细胞生物化学，尽管它们具有高毒性。在过去的几十年里，我们已经揭示了金属铜的金属转运体，其金属吸收和外排系统的总体，如何将环境中的金属含量和形态转化为细胞质金属交换。这是通过一种相当非特异性的广谱金属阳离子输入与外排系统对多余阳离子的特异性去除相结合实现的，外排系统有些从外周质输出底物，有些从细胞质输出底物。当对过渡金属的进口进行更深入的研究时，可以证明实际的进口途径会影响金属的分配效率，例如锌对细菌RNA聚合酶重要的RpoC （β -prime）亚基的分配。这表明在金莲中存在特定的金属输送通道和金属池，但吸收、外排、储存和特定分配机制之间的相互作用的大图景仍然没有框架。本文将对三种可能与锌结合的金属伴侣蛋白和gtp酶（CobW1至CobW3）在金属稳态中的作用进行表征，以了解锌离子的分配以及这三种CobW蛋白在这一过程中的作用。CobW1在缺锌条件下供应锌，CobW2可能是锌储存蛋白，是锌库的一部分，而CobW3不是GTPase，但可能与锌进口系统相互作用。与Uni Halle的Andrea Sinz合作，自下而上的方法将从生化和结构研究引导到交联质谱（XL-MS）相互作用组实验。这种自下而上的方法将首先通过等温量热法，重要构象的xml - ms导致结晶尝试或最重要的蛋白质构象的核磁共振研究（第一年）对三种CobW蛋白质进行详细的生化表征。在第二年，与选定的伙伴蛋白的相互作用将通过金属递送试验、生物核实验和xml - ms进行研究。第三年，选择不同条件下培养的金属双歧杆菌菌株，采用XL-MS进行相互作用研究。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踪实验来确定C. metallidurans中锌池的生化性质。

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.