NMR structure and metal interactions of the CopZ copper chaperone

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 funct ion is performed by the 8-kDa protein CopZ. This paper describes the NMR st ructure of apo-CopZ, obtained using uniformly N-15-labeled CopZ overexpress ed in Escherichia coli and NMR studies of the impact of Cu(I) binding on th e CopZ structure. The protein has a beta alpha beta beta alpha beta fold, w here the four beta-strands form an antiparallel twisted beta-sheet, and the two helices are located on the same side of the beta-sheet. A sequence mot if GMX-CXXC in the loop between the first beta-strand and the first a-helix contains the primary ligands, which bind copper(I), Binding of copper(I) c aused major structural changes in this molecular region, as manifested by t he fact that most NMR signals of the loop and the N-terminal part of the fi rst helix were broadened beyond detection. This effect was strictly localiz ed, because the remainder of the apo-CopZ structure was maintained after ad dition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared with apo-CopZ, ind icating 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 exc hangeable substrate rather than an integral part of the structure. Implicat ions of the present structural work for the in vivo function of CopZ are di scussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recog nition sites for other proteins that might be involved in copper transfer.