An NMR Study of the Interaction of the N-terminal Cytoplasmic Tail of the Wilson Disease Protein with Copper(I)-HAH1

Abstract

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 (ϳ630 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 ϳ630 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...

Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS)

“…This is a different behavior with respect to copper proteins that receive the metal from Atx1-like chaperones, which show the same fold and Cu(I)-binding site of hCCSD1. However, the partners of the Atx1-like chaperones are completely different from SOD1, not only in terms of overall fold but in terms of the metal coordination sites and their solvent accessibility (43)(44)(45)(46). hCCSD2, in addition to possessing the same fold of SOD1, contains a hydrophobic region of the β-barrel that is perfectly suited to interact with the hydrophobic region of the β-barrel of the SOD1 monomer.…”

Involvement of Protein Kinase D in Expression and Trafficking of ATP7B (Copper ATPase)

“…Even discrete mutations within the NMBD sequence were shown to interfere with targeting and Golgi retention of nascent ATP7B (24). In fact, the rather extended NMBD sequence is flexible and undergoes functionally relevant conformational interactions as shown by its structural resolution by NMR spectroscopy (25,26), its propensity to interact with chaperone proteins (27), and the influence of its copper site redox status on overall conformation (28). Finally, we found that mutation of the serine residues undergoing phosphorylation interfered strongly with formation of CTV, underscoring the role of PKD-sustained phosphorylation in the final trafficking step.…”

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

“…interactions proved to be difficult due to their transient nature and the largely independent motions of MBDs revealed by NMR studies (76).…”