ATP7B is a human P 1B -type ATPase that has a crucial role in maintaining copper(I) homeostasis. Mutations in the corresponding gene are the cause of Wilson disease. Among its various distinguishing features is a long (Ďł630 amino acids) N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We expressed the entire tail as a single construct in Escherichia coli and investigated its interaction with its copper chaperone (i.e. HAH1) by solution NMR spectroscopy. We observed that all six of the metal-binding domains were metallated by Cu(I)-HAH1, with the first, the second, and the fourth domains forming an adduct with it. This behavior is different from that of the highly similar human ATPase ATP7A, in which only two domains form such an adduct. The distinct behaviors of the different domains were analyzed in terms of the energetics of Cu(I) transfer, hinting at a specific role of the interaction with copper(I)-HAH1 in the overall functional process.Human ATP7B is a P 1B -type ATPase that, like the other human copper(I)-transporting ATPase, ATP7A, can translocate copper(I) to the trans-Golgi network, where the metal is incorporated into copper-dependent enzymes. Copper stimulation results in the redistribution of both ATP7A and ATP7B to the plasma membrane through intracellular vesicles in order to export excess copper out of the cytosol (1-4). Mutations in the ATP7B autosomal gene produce the copper accumulation in tissues that characterizes Wilson disease (5-7). Mutations in ATP7A are instead responsible for Menkes disease (8, 9). ATP7A and ATP7B are often referred to as the MNK and WLN proteins, respectively. P 1B -type ATPases contain four major regions/domains (the N-terminal copper-binding tail, the transmembrane domain, the ATP-binding domain, and the phosphatase domain) as well as a short C-terminal tail (10). The N-terminal copper-binding cytosolic tails of WLN and MNK are both Ďł630 amino acids long. They contain six 70-amino acid, independently folded metal-binding domains (MBDs), 2 which are relatively similar in sequence and structure (11)(12)(13)(14)(15). Each MBD harbors the conserved sequence motif GMXCXXC, through which it can bind one equivalent of copper(I) (16). The N-terminal tail has an important role in tuning the activity of the enzyme (17-19) and in modulating the intracellular trafficking rates of ATP7A and ATP7B (20 -22), an aspect of the cellular function of these ATPases that is lacking in less complex organisms, including yeast. The presence of either the intact fifth or intact sixth metal-binding domain in the human proteins is sufficient to support both WLN/MNK activity and intracellular trafficking at essentially normal levels (21,(23)(24)(25)(26). This has stimulated researchers to investigate the cellular role of the additional four domains that are present in the two human ATPases as well as in several other mammalian homologues. The K Cu binding affinity of the six MBDs of both the MNK (13, 27, 28) and WLN (29...