Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae

Division of Biochemistry, Freie Universität Berlin, Berlín, Berlin, Germany
JBIC Journal of Biological Inorganic Chemistry (Impact Factor: 2.54). 07/2004; 9(4):396-402. DOI: 10.1007/s00775-004-0536-1
Source: PubMed

ABSTRACT In Enterococcus hirae, copper homeostasis is controlled by the cop operon, which encodes the copper-responsive repressor CopY, the copper chaperone CopZ, and two copper ATPases, CopA and CopB. The four genes are under control of CopY, which is a homodimeric zinc protein, [Zn(II)CopY]2. It acts as a copper-responsive repressor: when media copper is raised, CopY is released from the DNA, allowing transcription to proceed. This involves the conversion of [Zn(II)CopY]2 to [Cu(I)2CopY]2, which is no longer able to bind to the promoter. Binding analysis of [Zn(II)CopY]2 to orthologous promoters and to control DNA by surface plasmon resonance analysis defined the consensus sequence TACAnnTGTA as the repressor binding element, or " cop box", of Gram-positive bacteria. Association and dissociation rates for the CopY-DNA interaction in the absence and presence of added copper were determined. The dissociation rate of [Zn(II)CopY]2 from the promoter was 7.3 x 10(-6) s(-1) and was increased to 5 x 10(-5) s(-1) in the presence of copper. This copper-induced change may be the underlying mechanism of copper induction. Induction of the cop operon was also assessed in vivo with a biosensor containing a lux reporter system under the control of the E. hirae cop promoter. Half-maximal induction of this biosensor was observed at 5 microM media copper, which delineates the ambient copper concentration to which the cop operon responds in vivo.

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Available from: Marc Solioz, Sep 25, 2015
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    • "In this scenario, the decreased growth phenotype observed in bacteria lacking CopA could be due to reduced copper availability to the cuproproteins of the periplasm. The presence in E. hirae of a soluble copper-chaperone CopZ and a copper-dependent transcription factor CopY [12,13] may also indicate that CopA is a component of a regulatory network that provides copper to the regulator molecules in a biologically available form. "
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    ABSTRACT: Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution.
    Current Genomics 04/2012; 13(2):124-33. DOI:10.2174/138920212799860661 · 2.34 Impact Factor
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    • "This leads to the replacement of the Zn 21 cofactor of CopY by two Cu 1 ions and a concomitant decrease in DNA affinity, which in turn induces the expression of the cop operon (Strausak & Solioz, 1997; Cobine et al., 2002). The transfer of copper from CopZ to CopY involves protein–protein interaction , thereby conferring specificity to the process (Cobine et al., 1999; Portmann et al., 2004). CopZ was also shown to interact with the CopA copper ATPase, the presumed entry point of copper into E. hirae (Multhaup et al., 2001). "
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    ABSTRACT: Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu(+) to the CopY repressor, thereby releasing its bound zinc and abolishing repressor-DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis.
    FEMS Microbiology Letters 10/2009; 302(1):69-75. DOI:10.1111/j.1574-6968.2009.01833.x · 2.12 Impact Factor
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    • "Functional assays based on geneknockout techniques can provide a useful method for checking the validity of mathematical models. In order to determine the rate constants for such models, various methods exist, such as surface plasmon resonance analysis (Slepak, 2000; Portmann et al., 2004 "
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    ABSTRACT: Calcium ions are the most ubiquitous and versatile signaling molecules in eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both the normal growth of the budding yeast Saccharomyces cerevisiae and the intricate working of the mammalian heart. In this paper, we make a detailed comparison between the calcium homeostasis/signaling networks in yeast cells and those in mammalian cardiac myocytes. This comparison covers not only the components, structure and function of the networks but also includes existing knowledge on the measured and simulated network dynamics using mathematical models. Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate similarly in mammalian cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is a simple organism that affords powerful genetic and genomic tools. Thus, exploring and understanding the calcium homeostasis/signaling system in yeast can provide a shortcut to help understand calcium homeostasis/signaling systems in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.
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