CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser46-containing β3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV–visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.
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Research Article|
April 14 2008
Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA
Chloe Singleton;
Chloe Singleton
*Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, U.K.
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Lucia Banci;
Lucia Banci
†Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy
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Simone Ciofi-Baffoni;
Simone Ciofi-Baffoni
†Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy
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Leonardo Tenori;
Leonardo Tenori
†Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy
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Margaret A. Kihlken;
Margaret A. Kihlken
*Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, U.K.
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Ruth Boetzel;
Ruth Boetzel
*Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, U.K.
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Nick E. Le Brun
Nick E. Le Brun
1
*Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, U.K.
1To whom correspondence should be addressed (email n.le-brun@uea.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 29 2007
Revision Received:
January 11 2008
Accepted:
January 23 2008
Accepted Manuscript online:
January 23 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 411 (3): 571–579.
Article history
Received:
November 29 2007
Revision Received:
January 11 2008
Accepted:
January 23 2008
Accepted Manuscript online:
January 23 2008
Citation
Chloe Singleton, Lucia Banci, Simone Ciofi-Baffoni, Leonardo Tenori, Margaret A. Kihlken, Ruth Boetzel, Nick E. Le Brun; Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA. Biochem J 1 May 2008; 411 (3): 571–579. doi: https://doi.org/10.1042/BJ20071620
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