Copperdependent Domaindomain Interactions And The Regulatory Role Of The Nterminal Domain

The N-terminal domain of human copper-transporting ATPases is essential for the copper-dependent functions of these proteins. Mutations of Cys residues in the metal-binding motifs inactivate the copper-transporting activity of WNDP and prevent copper-dependent trafficking of WNDP in a cell (34,45). At the same time, recent studies conducted in several laboratories convincingly demonstrated that the entire N-terminal domain was not essential for the transport function of copper-ATPases: the large portion of this domain could be deleted or mutagenized without significant loss of the copper-transport activity (34,43,44). These results are consistent with the fact that the bacterial, yeast, and plant copper-ATPase can carry out their functions with fewer than six (one to three) metal-binding repeats (e.g., see ref. 62). Because extra metal-binding repeats are not important for function, it seems likely that they play a role in regulation of WNDP and MNKP.

Fig. 5. Hypothetical model showing how ATP and ADP could bind to ATP-BD simultaneously (55) in the isolated ATP-BD (see text for details).

The regulatory role for the N-terminal domain has been suggested by several authors (20,44,48,53), and recent studies indicate that copper binding to the N-terminal domain triggers the intracellular relocalization of MNKP and WNDP (44,45). However, only one or two metal-binding repeats, which are important for transport function, seem to be necessary and sufficient for copper-induced trafficking (44,63,64). Therefore, the first four metal-binding repeats were proposed to function by preventing protein trafficking before they are filled up with copper (see the model in ref. 53).

Our recent studies shed some light on how the N-terminal domain may regulate the copper-dependent functions of WNDP. We found that N-WNDP interacts specifically with the ATP-binding domain of WNDP and that the interactions between these two domains are copper dependent: In the absence of copper, two domains interact tightly, whereas copper-bound N-WNDP does not bind to ATP-BD very well (55). How many bound copper atoms are sufficient to induce the change in intradomain interactions is a subject of future investigations.

Importantly, the domain-domain interactions have a clear effect on the conformational state of the ATP-binding domain: when N-WNDP is bound to ATP-BD, the affinity of the latter to nucleotide is decreased several-fold (see Table 1). Therefore it is tempting to speculate that binding of copper to the N-terminal domain is accompanied by conformational changes that alter the interaction between N-WNDP and the ATP-binding domain (Fig. 6). This change, in turn, may modify the nucleotide-binding properties of WNDP and possibly the rate of ATP-hydrolysis.

It is also quite possible that copper-induced changes in domain-domain interactions and the subsequent conformational transitions lead to exposure of sites for the intracellular trafficking machinery, resulting in copper-dependent relocalization of WNDP in a cell. The mutagenesis studies and analysis of the naturally occurring mutants (63,65) revealed that the copper-dependent trafficking of MNKP and WNDP can be disrupted by amino acid substitutions in various regions of these proteins. These results suggest that a certain conformation of a functionally active protein rather than its mere ability to bind copper is important for trafficking of copper-ATPase. Although the precise molecular mecha-

Fig. 6. Copper binding to N-WNDP decreases the interactions of N-terminal domain with ATP-BD, which in turn, changes the ATP-BD conformation.

nism of the WNDP trafficking and targeting is still unclear, it is likely to involve series of copper-dependent posttranslational events, based on changes in domains conformation induced by copper.


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