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

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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.