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Distinct characteristics of Ag+ and Cd2+ binding to CopZ from Bacillus subtilis

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Abstract

The chaperone CopZ together with the P-type ATPase transporter CopA constitute a copper-detoxification system in Bacillus subtilis that is commonly found in bacteria and higher cells. Previous studies of the regulation of the copZA operon showed that expression is significantly upregulated in response to elevated concentrations of environmental silver and cadmium, as well as copper. Here, we have used spectroscopic and bioanalytical methods to investigate in detail the capacity of CopZ to bind these metal ions (as Ag+ and Cd2+). We demonstrate that Ag+ binding mimics closely that of Cu+: Ag+-mediated dimerisation of the protein occurs, and distinct Ag+-bound species are formed at higher Ag+ loadings. Cd2+ also binds to CopZ, but exhibits significantly different behaviour. Cd2+-mediated dimerisation is only observed at low loadings, such that at 0.5 and one Cd2+ per CopZ the protein is present mainly in a monomeric form; and multinuclear higher-order forms of Cd2+–CopZ are not observed. Competition binding studies reveal that Ag+ binds with an affinity very similar to that of Cu+, while Cd2+ binding is significantly weaker. These data provide support for the proposal that CopZ may be involved in the detoxification of silver and cadmium, in addition to copper.

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Abbreviations

BCS:

Bathocuproine disulfonate

CD:

Circular dichroism

ICP-AES:

Inductively coupled plasma atomic emission spectroscopy

LMCT:

Ligand to metal charge transfer

Mops:

3-Morpholinopropanesulfonate

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Acknowledgments

We thank the BBSRC for the award of studentships to C.S. and M.A.K., and the Wellcome Trust for an award from the Joint Infra-structure Fund for equipment. We are grateful to Tom Clarke and David Richardson for assistance with analytical ultracentrifugation experiments (the analytical ultracentrifugation facility at the University of East Anglia was funded by the Wellcome Trust and HEFCE) and Myles Cheesman and Andrew Thomson for access to spectroscopic instrumentation.

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Authors and Affiliations

  1. Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK

    Margaret A. Kihlken, Chloe Singleton & Nick E. Le Brun

Authors
  1. Margaret A. Kihlken
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  2. Chloe Singleton
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  3. Nick E. Le Brun
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Correspondence to Nick E. Le Brun.

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Margaret A. Kihlken and Chloe Singleton contributed equally to this work.

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Kihlken, M.A., Singleton, C. & Le Brun, N.E. Distinct characteristics of Ag+ and Cd2+ binding to CopZ from Bacillus subtilis . J Biol Inorg Chem 13, 1011–1023 (2008). https://doi.org/10.1007/s00775-008-0388-1

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