CopYDNA complex

DNA only

Fig. 9. Effect of Cu(I)CopZ on the CopY-DNA interaction. Native CopY retarded a 32P-labeled oligonucleotide promoter fragment in the band-shift assay. Cu(I)CopZ abolished binding of CopY to the promoter, but not Cu(I)MNKr4. The artificial copper(I) complex Cu(I)acetonitrile could also donate copper to CopY.

CopY (43). These findings were further supported by quantitative gel filtration chromatography, paired with metal analysis.

4.2. Solution Structure of CopZ

To further study the chaperoning function by CopZ, its three-dimensional structure was determined. Universally 15N-labeled CopZ was overexpressed in E. coli and purified to homogeneity. By nuclear magnetic resonance (NMR) spectroscopy, the solution structures of apo-CopZ and Cu(I)CopZ were derived (44). The structure of apo-CopZ was very well defined: the root mean square deviation (r.m.s.d.) of the backbone heavy atoms within the secondary structure elements was 0.32 A. The bundle of the 20 best conformers is shown in Fig. 10. The amino acid chain in CopZ adopts a PaPPaP fold. The P-strands form an antiparallel P-sheet that is strongly twisted. The two a-helices are packed against the P-sheet. They enclose an angle of about 45°. Figure 11 shows a ribbon drawing of the CopZ molecule. The two copper-binding residues Cys-11 and Cys-14 are located in the loop that connects the first P-strand with the first a-helix.

The charged side chains on the surface of the protein are distributed very unevenly, so that large negatively and positively charged patches exist on the protein surface. The global fold is essentially identical to that of the mercury-binding protein MerP (45), mbd4, the fourth metal-binding domain of the Menkes copper-transporting ATPase (39), Atx1, the yeast analogon to CopZ (46), and Hah1, the human analogon to both CopZ and Atx-1 (47). A detailed comparison of the structure of CopZ with those of mbd4 and MerP shows that the structures are nearly identical except for the metal-binding loop, where the CxxC motif is located. The relative conformations of Cys-11 and Cys-14 in CopZ are such that metal binding by both of them requires structural rearrangement (see Fig. 12). This is clearly not the case in mbd4, which can accommodate Ag(I) apparently without any changes in structure. In MerP, only the loop between Pj and aj is rearranging upon Hg(II)-binding whereas in CopZ, it seems that the first a-helix is taking part in the required rearrangement. This difference in behavior might be the result of the presence of two prolines flanking the metal-binding loop in MerP and may be preventing

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