Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.     

A NMR study of the interaction of a three-domain construct of ATP7A with copper(I) and copper(I)-HAH1: the interplay of domains

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal tail protruding into the cytosol and containing six copper(I)-binding domains, which are individually folded and capable of binding one copper(I) ion. ATP7A receives copper from a soluble protein, the metallochaperone HAH1. The exact role and interplay of the six soluble domains is still quite unclear, as it has been extensively demonstrated that they are strongly redundant with respect to copper(I) transport in vivo. In the present work, a three-domain (fourth to sixth, MNK456) construct has been investigated in solution by NMR, in the absence and presence of copper(I). In addition, the interaction of MNK456 with copper(I)-HAH1 has been studied. It is proposed that the fourth domain is the preferential site for the initial interaction with the partner. A significant dependence of the overall domain dynamics on the metallation state and on the presence of HAH1 is observed. This dependence could constitute the molecular mechanism to trigger copper(I) translocation and/or ATP7A relocalization from the trans-Golgi network to the plasmatic membrane.     

Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase.

The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.     

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.     

Mechanisms of the copper-dependent turnover of the copper chaperone for superoxide dismutase

The copper chaperone for superoxide dismutase (CCS) is an intracellular metallochaperone required for incorporation of copper into the essential antioxidant enzyme copper/zinc superoxide dismutase (SOD1). Nutritional studies have revealed that the abundance of CCS is inversely proportional to the dietary and tissue copper content. To determine the mechanisms of copper-dependent regulation of CCS, copper incorporation into SOD1 and SOD1 enzymatic activity as well as CCS abundance and half-life were determined after metabolic labeling of CCS-/- fibroblasts transfected with wild-type or mutant CCS. Wild-type CCS restored SOD1 activity in CCS-/- fibroblasts, and the abundance of this chaperone in these cells was inversely proportional to the copper content of the media, indicating that copper-dependent regulation of CCS is entirely post-translational. Although mutational studies demonstrated no role for CCS Domain I in this copper-dependent regulation, similar analysis of the CXC motif in Domain III revealed a critical role for these cysteine residues in mediating copper-dependent turnover of CCS. Further mutational studies revealed that this CXC-dependent copper-mediated turnover of CCS is independent of the mechanisms of delivery of copper to SOD1 including CCS-SOD1 interaction. Taken together these data demonstrate a mechanism determining the abundance of CCS that is competitive with the process of copper delivery to SOD1, revealing a unique post-translational component of intracellular copper homeostasis.     

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.     

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.     

Cu(I) binding and transfer by the N terminus of the Wilson disease protein

Wilson and Menkes diseases are genetic disorders of copper metabolism caused by mutations in the Wilson (WND) and Menkes (MNK) copper-transporting P1B-type ATPases. The N termini of these ATPases consist of six metal binding domains (MBDs). The MBDs interact with the copper chaperone Atox1 and are believed to play roles in catalysis and in copper-mediated cellular relocalization of WND and MNK. Although all six MBDs have similar folds and bind one Cu(I) ion via a conserved CXXC motif, biochemical and genetic data suggest that they have distinct functions. Most studies aimed at characterizing the MBDs have employed smaller polypeptides consisting of one or two domains. The role of each MBD is probably defined by its environment within the six-domain N terminus, however. To study the properties of the individual domains within the context of the intact Wilson N terminus (N-WND), a series of variants in which five of the six metal binding CXXC motifs are mutated to SXXS was generated. For each variant, the Cu(I) binding affinity and the ability to exchange Cu(I) with Atox1 were investigated. The results indicate that Atox1 can deliver Cu(I) to and remove Cu(I) from each MBD, that each MBD has stronger Cu(I) retention properties than Atox1, and that all of the MBDs as well as Atox1 have similar K(Cu) values of (2.2-6.3) x 10(10) m(-1). Therefore, the specific role of each MBD is not conferred by its position within the intact N-WND but may be related to interactions with other domains and partner proteins.     

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.     

A gain of superoxide dismutase (SOD) activity obtained with CCS, the copper metallochaperone for SOD1

The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp(200)). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS.     

X-ray absorption spectroscopy of the copper chaperone HAH1 reveals a linear two-coordinate Cu(I) center capable of adduct formation with exogenous thiols and phosphines

The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm. The implications of these findings for mechanisms of copper transport are discussed.     

Crystal structure of the copper chaperone for superoxide dismutase.

Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins. The 1.8 A resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains. The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake. The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis. In the crystal, two SOD1-like domains interact to form a dimer. The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone.     

Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2

The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.     

Transduced human copper chaperone for Cu,Zn-SOD (PEP-1-CCS) protects against neuronal cell death

Reactive oxygen species (ROS) contribute to the development of various human diseases. Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS. SOD activity is dependent upon bound copper ions supplied by its partner metallochaperone protein, copper chaperone for SOD (CCS). In the present study, we investigated the protective effects of PEP-1-CCS against neuronal cell death and ischemic insults. When PEP-1-CCS was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Moreover, transduced PEP-1-CCS markedly increased endogenous SOD activity in the cells. Immunohistochemical analysis revealed that it prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that CCS is essential to activate SOD, and that transduction of PEP-1-CCS provides a potential strategy for therapeutic delivery in various human diseases including stroke related to SOD or ROS.     

Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

CopA, a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1-like domains separated by a short linker at its N-terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans, 688-696]. To try to understand the contributions of each domain to the complex Cu(I)-binding behaviour of this and related proteins, we purified a wild-type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity (K = ～ 1 × 10(18) m(-1) ) and underwent Cu(I)-mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid-base properties revealed pK(a) values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct.     

Heterodimeric structure of superoxide dismutase in complex with its metallochaperone

The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.     

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.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.