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: 3.16). 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, Aug 04, 2015
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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 · 2.72 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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    • "ehCopY) a homodimeric, copper inducible repressor. EhCopY regulates the expression of the whole operon (Odermatt et al. 1992; Odermatt et al. 1994; Odermatt & Solioz 1995; Solioz & Stoyanov 2003). In low copper media, ehCopY is present as a Zn(II) containing homodimer and is bound to the operator/promoter of the cop operon (Strausak & Solioz 1997; Portmann et al. BioMetals (2006) 19: 61–70 Ó Springer 2006 DOI 10.1007/s10534-005-5381-3 2004). There are two binding sites, each one interacting with a ehCopY dimer. These ehCopY binding sites feature the inverted repeat TAC- AnnTGTA, called the 'cop box', which appears to be a conserved motif in copper regulation among Firmicutes. When the copper concentration in E. hirae is elevated, the copper-loaded chaperone, Cu(I)CopZ, tr"
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    ABSTRACT: CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.
    BioMetals 03/2006; 19(1):61-70. DOI:10.1007/s10534-005-5381-3 · 2.69 Impact Factor
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