An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.     

The different intermolecular interactions of the soluble copper-binding domains of the menkes protein, ATP7A

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal cytosolic tail containing six domains that are individually folded and capable of binding one copper(I) ion each. We investigated the entire N-terminal tail (MNK1-6) in solution by NMR spectroscopy and addressed its interaction with copper(I) and with copper(I)-HAH1, the physiological partner of ATP7A. At copper(I)-HAH1:MNK1-6 ratios of up to 3:1, thus encompassing the range of protein ratios in vivo, both the first and fourth domain of the tail formed a metal-mediated adduct with HAH1 whereas the sixth domain was simultaneously able to partly remove copper(I) from HAH1. These processes are not dependent on one another. In particular, formation of the adducts is not necessary for copper(I) transfer from HAH1 to the sixth domain. The present data, together with available in vivo studies, suggest that the localization of ATP7A between the trans-Golgi network and the plasma membrane may be regulated by the accumulation of the adducts with HAH1, whereas the main role of domains 5 and 6 is to assist copper(I) translocation.     

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.     

An atomic-level investigation of the disease-causing A629P mutant of the Menkes protein, ATP7A

Menkes disease is a fatal disease that can be induced by various mutations in the ATP7A gene, leading to unpaired uptake of dietary copper. The ATP7A gene encodes a copper(I)-translocating ATPase. Here the disease-causing A629P mutation, which occurs in the last of the six copper(I)-binding soluble domains of the ATPase (hereafter MNK6), was investigated. To understand why this apparently minor amino acid replacement is pathogenic, the solution structures and dynamics on various time-scales of wild-type and A629P-MNK6 were determined both in the apo- and copper(I)-loaded forms. The interaction in vitro with the physiological ATP7A copper(I)-donor (HAH1) was additionally studied. The A629P mutation makes the protein beta-sheet more solvent accessible, possibly resulting in an enhanced susceptibility of ATP7A to proteolytic cleavage and/or in reduced capability of copper(I)-translocation. A small reduction of the affinity for copper(I) is also observed. Both effects could concur to pathogenicity.     

Metal binding domains 3 and 4 of the Wilson disease protein: solution structure and interaction with the copper(I) chaperone HAH1

The Wilson disease protein or ATP7B is a P 1B-type ATPase involved in human copper homeostasis. The extended N-terminus of ATP7B protrudes into the cytosol and contains six Cu(I) binding domains. This report presents the NMR structure of the polypeptide consisting of soluble Cu(I) binding domains 3 and 4. The two domains exhibit ferredoxin-like folds, are linked by a flexible loop, and act independently of one another. Domains 3 and 4 tend to aggregate in a concentration-dependent manner involving nonspecific intermolecular interactions. Both domains can be loaded with Cu(I) when provided as an acetonitrile complex or by the chaperone HAH1. HAH1 forms a 70% complex with domain 4 that is in fast exchange with the free protein in solution. The ability of HAH1 to form a complex only with some domains of ATP7B is an interesting property of this class of proteins and may have a signaling role in the function of the ATPases.     

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.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

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.     

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.     

Interaction of the two soluble metal-binding domains of yeast Ccc2 with copper(I)-Atx1

Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second. Similar results are found in ATPases from other organisms and may represent a general feature, whose biochemical implications are not yet fully appreciated.     

Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl beta-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 microM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 microM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.     

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.     

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.     

Solution structure of the apo and copper(I)-loaded human metallochaperone HAH1

The human metallochaperone HAH1 has been produced in Escherichia coli with four additional amino acids at the C-terminus and characterized in solution by NMR spectroscopy, both with and without copper(I). The solution structure of the apo-HAH1 monomer has a root-mean-square-deviation (RMSD) of 0.50 A for the coordinates of the backbone atoms and 0.96 A for all heavy atoms. These values compare, respectively, with 0.45 and 0.95 A for copper(I)-HAH1. There are only minor structural rearrangements upon copper(I) binding. In particular, the variation of interatomic interactions around the metal-binding region is limited to a movement of Lys60 toward the metal site. The protein structures are similar to those obtained by X-ray crystallography in a variety of derivatives, with backbone RMSD values below 1 A. In the holoprotein, copper(I) is confirmed to be two coordinated. If these data are compared with those of orthologue proteins, we learn that HAH1 has a lower tendency to change coordination number from two to three. Such a switch in coordination is a key step in copper transfer.     

Solution structure and backbone dynamics of the Cu(I) and apo forms of the second metal-binding domain of the Menkes protein ATP7A

The second domain of the human Menkes protein (MNK2), formed by 72 residues, has been expressed in Escherichia coli, and its structure has been determined by NMR in both the apo and copper-loaded forms. The structures, obtained with (13)C- and (15)N-labeled samples, are of high quality with backbone rmsd values of 0.51 and 0.41 A and CYANA target functions of 0.39 and 0.38 A(2), respectively. The loop involved in copper binding is part of a hydrophobic patch, which is maintained in both forms. Conformational mobility is observed in the apo form in the same loop. A comparison with metallochaperones and soluble domains of P-type ATPases allows us to relate the primary structure to the occurrence of structural rearrangements upon copper binding.     

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 .     

Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains

BackgroundA copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCS^dI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCS^dI in CCS.MethodsTo evaluate protein-protein interactions of CCS through CCS^dI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed.ResultsWe found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCS^dI. The interaction between CCS^dI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCS^dI and HAH1.ConclusionWhile physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCS^dI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.     

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.     

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230Asp, within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.     

At sixes and sevens: cryptic domain in the metal binding chain of the human copper transporter ATP7A

ATP7A and ATP7B are structurally similar but functionally distinct active copper transporters that regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. Both proteins have a chain of six cytosolic metal-binding domains (MBDs) believed to be involved in the copper-dependent regulation of the activity and intracellular localization of these enzymes. Although all the MBDs are quite similar in structure, their spacing differs markedly between ATP7A and ATP7B. We show by NMR that the long polypeptide between MBD1 and MBD2 of ATP7A forms an additional seventh metastable domain, which we called HMA1A (heavy metal associated domain 1A). The structure of HMA1A resembles the MBDs but contains no copper-binding site. The HMA1A domain, which is unique to ATP7A, may modulate regulatory interactions between MBD1–3, contributing to the distinct functional properties of ATP7A and ATP7B.