NMR studies of the soluble metal binding domains of the copper transport ATPase from B-subtilis

Regulation of intracellular copper activity is crucially important for cell life. Copper is very toxic to both eukaryotic and prokaryotic cells as it is involved in oxidative reactions with the production of radicals. In bacteria the mechanisms of copper homeostasis are poorly understood. CopA, a P-type membrane bound ATPase of B. subtilis, is apparently involved in copper binding and transport; it contains two cysteines in each of two putative soluble N-terminal metal binding domains. In this work the structural characterization of the two soluble domains has been performed by NMR. Complete resonances only for the second domain can be detected while the first one shows only signals for a few residues. The same behavior is observed also for the individual, isolated domains, indicating a not complete folding for the first domain. Atomic absorption determination revealed a 1:1 copper/protein ratio. The goal of this work is to investigate the influence of copper binding in the protein structure, in order to better understand the mechanism of copper homeostasis in bacteria.