N-TERMINAL DOMAINS OF HUMAN COPPER-TRANSPORTING ADENOSINE TRIPHOSPHATASES (THE WILSONS AND MENKES DISEASE PROTEINS) BIND COPPER SELECTIVELYIN-VIVO AND IN-VITRO WITH STOICHIOMETRY OF ONE COPPER PER METAL-BINDING REPEAT

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions;vith maltose-binding protein, purified, and their metal-bindi ng properties were characterized, Both N-MNK and N-WND bind copper spe cifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues hi the presence of different heavy metals. When E. coil cells are grown in the presence of copper, N-MNK and N-WND bind copper in v ivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper r eleased from the copper-N-MNK and copper-N-WND complexes reacts with t he Cu(I)-selective chelator bicinchoninic acid in the absence of reduc ing agents. This suggests that in proteins, it is bound in reduced Cu( I) form, in agreement with the spectroscopic properties of the copper- bound domains. Copper bound to the domains in vivo or in vitro specifi cally protects the N-MNK and N-WND against labeling with the cysteine- directed probe; this indicates that Cys residues in the repetitive mot ifs GMTCXPCXXXIE are involved in coordination of copper. Direct involv ement of the N-terminal domains in the binding of copper suggests thei r important role in copper-dependent functions of human copper-transpo rting adenosine triphosphatases (Wilson's and Menkes disease proteins) .