Copper Trafficking:  the Solution Structure of Bacillus subtilis CopZ

Abstract

A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed. The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions. The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample. A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms. NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure. Some disorder is observed in the loop devoted to copper binding. These results are compared with those reported for CopZ from E. hirae whose structure is well-defined only in the apo form. The different behaviors of copper-loaded E. hirae and B. subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form. The comparison is extended to other similar proteins, with particular attention to the copper-binding loop. The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process. Proposals for the role of CopZ are also presented.

Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

“…MBDs are 60-to 70-aa-long domains. They have a ␤␣␤␤␣␤ fold and an invariant CXXC metal-binding sequence similar to the well described Cu ϩ chaperones, Atox1, Atx1, and CopZ (20,22,23). N-MBDs bind Cu ϩ with high affinity (24)(25)(26), and in vivo, they receive Cu ϩ from the corresponding Cu ϩ chaperones (23,27,28).…”

“…However, Cu ϩ does not access the ATPase as a free (hydrated) ion but is bound to a chaperone protein. Cu ϩ chaperones that deliver metal specifically to Cu ϩ -ATPases have been identified in a range of organisms (4,20,22). We recently described the A. fulgidus Cu ϩ chaperone CopZ (32).…”

Conserved residues modulate copper release in human copper chaperone Atox1

“…1A). Structural work has demonstrated that Cu chaperones and target metal-binding domains from various organisms possess the same fold and coordinate the metal via two surfaceexposed cysteines in the MXCXXC motif (13)(14)(15)(16)(17)(18)(19)(20)(21). Although the methionine in the first position of the MXCXXC motif is conserved in all organisms, it is not directly involved in metal ligation (13).…”

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

“…The Atox1 metallochaperone and its homologs, both eukaryotic and prokaryotic, contain a single CXXC motif (14). Crystal and solution structures of Atox1 (15), yeast Atx1 (16,17), bacterial CopZ (18,19), and single domains of MNK (20) and Ccc2 (21) reveal a conserved ␤␣␤␤␣␤-fold with the cysteines from the CXXC motif coordinating metal ions on a surface-exposed loop. Notably, the CXXC motifs from two Atox1 molecules coordinate a single Cu(I) ion in the x-ray structure (15).…”