NMR Structure and Metal Interactions of the CopZ Copper Chaperone

Abstract

A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. In Enterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly 15 N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a ␤␣␤␤␣␤ fold, where the four ␤-strands form an antiparallel twisted ␤-sheet, and the two helices are located on the same side of the ␤-sheet. A sequence motif GMX-CXXC in the loop between the first ␤-strand and the first ␣-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared with apo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer.

Yeast Cox17 Solution Structure and Copper(I) Binding

“…Crystal and solution structures of Atx1 (50, 51), Atox1 (52), a bacterial homolog called CopZ (53,54), and domains of their target copper transport ATPases (55)(56)(57) reveal that the metal-binding site is located on a surface-exposed loop between the first ␤ strand and the first ␣ helix in the ␤␣␤␤␣␤ fold, with the first cysteine derived from the loop and the second cysteine derived from the N terminus of the helix. Similarly, the copper-binding site in Cox17 is located on a loop with the second coordinating cysteine at the N terminus of a helix (Fig.…”

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

“…The fourth of the six metal binding domains of the human Menkes copper ATPase and the first copper binding domain of the yeast Ccc2 copper ATPase possess essentially the same structure (Gitschier et al 1998). Interestingly, these domains exhibit the same structure as the small chaperones which route copper intracellularly, like CopZ of E. hirae, Atx1 of Saccharomyces cerevisiae, or human Hah1 (Wimmer et al 1999;Rosenzweig et al 1999;Wernimont et al 2000). The structures of the A-domain of the CopA copper ATPase of the thermophile Archaeoglobus fulgidus (Sazinsky et al 2006a) as well as the combined PN-domains of A. fulgidus (Sazinsky et al 2006b) and Sulfolobus solfataricus (Lübben et al 2007) have also recently been solved.…”

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).…”