Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.     

Binding of copper(I) by the Wilson disease protein and its copper chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.     

Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.     

The multi-layered regulation of copper translocating P-type ATPases

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

Copper transfer to the N-terminal domain of the Wilson disease protein (ATP7B): X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands

The copper-transporting ATPases are 165-175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu-Cu interaction which may signal a conformational change of the N-terminal domain.     

Distinct functions of serial metal‐binding domains in the Escherichia coli P1B‐ATPase CopA

P_1B‐ATPases are among the most common resistance factors to metal‐induced stress. Belonging to the superfamily of P‐type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P_1B‐ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N‐terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal‐binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein‐internal pathway of copper and demonstrate the distal N‐terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane‐integral ion‐binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P_1B‐ATPases, which is governed by the length of the inter‐domain linker.     

Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics

After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.     

Differential regulation of the Menkes and Wilson disease copper transporters by hormones: an integrated model of metal transport in the placenta

Copper (Cu) plays a critical role in the developing foetus, but virtually nothing is known concerning the regulation of its uptake and metabolism in the placenta. In this issue of the Biochemical Journal, Hardman and colleagues, using a model of placental trophoblasts in culture, identify differential hormonal regulation of two copper-transporting ATPases; namely, those responsible for Menkes disease (ATP7A; MNK) and Wilson disease (ATP7B; WND). Insulin and oestrogen, which are essential during gestation, up-regulate MNK and this leads to trafficking of the MNK protein from the Golgi to the basolateral membrane, resulting in increased Cu efflux. At the same time, insulin decreased WND levels, and this leads to intracellular sequestration of the protein to a perinuclear region that reduces apical Cu release. As such, this results in a concerted flux of Cu from the basolateral surface of the trophoblast that would potentially be used by the developing foetus. An integrated model of vectorized Cu transport is proposed, which involves co-ordinated expression of transporters, organelle interactions and probable protein-protein interactions. The findings have wider implications for considering general models of intracellular metal transport.     

Characterisation of copper-binding to the second sub-domain of the Menkes protein ATPase (MNKr2)

The Menkes ATPase (MNK) has an essential role in the translocation of copper across cellular membranes. In a complementary manner, the intracellular concentration of copper regulates the activity and cellular location of the ATPase through its six homologous amino-terminal domains. The roles of the six amino-terminal domains in the activation and cellular trafficking processes are unknown. Understanding the role of these domains relies on the development of an understanding of their metal-binding properties and structural properties. The second conserved sub-domain of MNK was over-expressed, purified and its copper-binding properties characterised. Reconstitution studies demonstrate that copper binds to MNKr2 as Cu(I) with a stoichiometry of one copper per domain. This is the first direct evidence of copper-binding to the MNK amino-terminal repeats. Circular dichroism studies suggest that the binding or loss of copper to MNKr2 does not cause substantial changes to the secondary structure of the protein.     

Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein

The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.     

Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.     

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.     

Expression, purification and copper-binding studies of the first metal-binding domain of Menkes protein.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.     

A comparison of the mutation spectra of Menkes disease and Wilson disease

The genes for two copper-transporting ATPases, ATP7A and ATP7B, are defective in the heritable disorders of copper imbalance, Menkes disease (MNK) and Wilson disease (WND), respectively. A comparison of the two proteins shows extensive conservation in the signature domains, with amino acid identities outside of the conserved domains being limited. The mutation spectra of MNK and WND were compared to confirm and refine further regions critical for normal function. Mutations were found to be relatively widespread; however, the majority was concentrated within defined functional domains and membrane-spanning segments, reinforcing the importance of these regions for protein function. Of the total published point mutations in ATP7A, 23.0% are splice-site, 20.7% nonsense, 17.2% missense, and 39.1% small insertions/deletions. There is a high prevalence (58.2%) of missense mutations in ATP7B. For the other mutations in ATP7B, 7.4% are splice-site, 7.4% nonsense, and 27.0% small insertions/deletions. A region of possible importance is the intervening sequence between the last copper-binding domain and the first transmembrane helix, as this region has a high percentage of MNK mutations. Similarly, the region containing the ATP-binding domain has 24.6% of all WND mutations. The study of mutation locations is useful for defining critical regions or residues and for efficient molecular diagnosis.Electronic Supplementary Material Supplementary material is available for this article if you access the article at .     

Molecular mechanisms of copper metabolism and the role of the Menkes disease protein

Menkes disease is an X‐linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births. The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood. The symptoms of Menkes disease result from a deficiency of serum copper and copper‐dependent enzymes. A candidate gene for the disease has been isolated and designated MNK. The MNK gene codes for a P‐type cation transporting ATPase, based on homology to known P‐type ATPases and in vitro experimentation. cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments. The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal‐binding repeats in the amino‐terminal domain. The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease. Each of the Menkes protein amino‐terminal repeats contains a conserved ‐X‐Met‐X‐Cys‐X‐X‐Cys‐ motif (where X is any amino acid). These metal‐binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes. An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented. This forms the basis for a discussion of Menkes disease and the protein deficit in this disease. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 93–106, 1999     

Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein.

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.     

Characterization of the interaction between the Wilson and Menkes disease proteins and the cytoplasmic copper chaperone, HAH1p.

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex.