Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA.
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 Ser(46)-containing beta3-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.
Singleton
Chloe
C
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
Banci
Lucia
L
Ciofi-Baffoni
Simone
S
Tenori
Leonardo
L
Kihlken
Margaret A
MA
Boetzel
Ruth
R
Le Brun
Nick E
NE
D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012995:Solubility; D013053:Spectrophotometry
D001059:Apoproteins; D001426:Bacterial Proteins; C071263:CopA protein, Bacteria; D003300:Copper