The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites.

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ∼2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed.

Levofloxacin reduces inflammatory cytokine levels in human bronchial epithelia cells: implications for aerosol MP-376 (levofloxacin solution for inhalation) treatment of chronic pulmonary infections.

Inflammation resulting from chronic bacterial infection in the lung contributes to long-term pulmonary complications in chronic pulmonary infections such as cystic fibrosis. Aerosol administration of levofloxacin as in the form of the investigational formulation MP-376 results in higher concentrations in lung tissues that are higher than those that can be attained with oral or intravenous dosing of levofloxacin. The objective of this study was to evaluate the effect of high concentrations of levofloxacin achieved with aerosol administration of MP-376 on proinflammatory cytokine secretion by immortalized human bronchial epithelia cells in vitro. Additionally, we investigated the potential mechanisms of the immunomodulatory effect of levofloxacin. In vitro studies in human lung epithelial cell lines showed that levofloxacin led to a dose-related reduction in IL-6 and IL-8 concentrations, with 300 µg mL(-1) resulting in the reduction of levels of IL-6 by fourfold and IL-8 by twofold (P<0.05); in contrast, tobramycin increased IL-6 levels by 50%, but had no effect on IL-8. Levofloxacin treatment did not affect the cytokine mRNA level and nuclear factor-κB-dependent promoter activity. These findings suggest that high concentrations of levofloxacin obtained in pulmonary tissues following the administration of aerosol MP-376 may provide additional benefits in patients with chronic pulmonary infections that are independent of its antibacterial properties.

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes.

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.

Solution structure of the N-domain of Wilson disease protein: distinct nucleotide-binding environment and effects of disease mutations.

Wilson disease protein (ATP7B) is a copper-transporting P(1B)-type ATPase that regulates copper homeostasis and biosynthesis of copper-containing enzymes in human tissues. Inactivation of ATP7B or related ATP7A leads to severe neurodegenerative disorders, whereas their overexpression contributes to cancer cell resistance to chemotherapeutics. Copper-transporting ATPases differ from other P-type ATPases in their topology and the sequence of their nucleotide-binding domain (N-domain). To gain insight into the structural basis of ATP7B function, we have solved the structure of the ATP7B N-domain in the presence of ATP by using heteronuclear multidimensional NMR spectroscopy. The N-domain consists of a six-stranded beta-sheet with two adjacent alpha-helical hairpins and, unexpectedly, shows higher similarity to the bacterial K(+)-transporting ATPase KdpB than to the mammalian Ca(2+)-ATPase or Na(+),K(+)-ATPase. The common core structure of P-type ATPases is retained in the 3D fold of the N-domain; however, the nucleotide coordination environment of ATP7B within this fold is different. The residues H1069, G1099, G1101, I1102, G1149, and N1150 conserved in the P(1B)-ATPase subfamily contribute to ATP binding. Analysis of the frequent disease mutation H1069Q demonstrates that this mutation does not significantly affect the structure of the N-domain but prevents tight binding of ATP. The structure of the N-domain accounts for the disruptive effects of >30 known Wilson disease mutations. The unique features of the N-domain provide a structural basis for the development of specific inhibitors and regulators of ATP7B.

The copper-transporting ATPases, menkes and wilson disease proteins, have distinct roles in adult and developing cerebellum.

Copper is essential for brain metabolism, serving as a cofactor to superoxide dismutase, dopamine-beta-hydroxylase, amyloid precursor protein, ceruloplasmin, and other proteins required for normal brain function. The copper-transporting ATPases ATP7A and ATP7B play a central role in distribution of copper in the central nervous system; genetic mutations in ATP7A and ATP7B lead to severe neurodegenerative disorders, Menkes disease and Wilson disease, respectively. Although both ATP7A and ATP7B are required, their specific roles and regulation in the brain remain poorly understood. Using high-resolution imaging and functional assays, we demonstrate that ATP7A and ATP7B show cell-specific distribution in adult cerebellum, have distinct enzymatic characteristics, and are regulated differently during development. ATP7B is continuously expressed in Purkinje neurons (PN) where it delivers copper to the ferroxidase ceruloplasmin. ATP7A is a faster copper transporter than Wilson disease protein as evidenced by faster rates of catalytic reactions. The expression of ATP7A switches during development from PN to Bergmann glia, the cells supporting PN function in adult brain. Inactivation of ATP7B (Wilson disease protein) by gene knock-out induces a striking shift in the expression of the ATP7B target protein, ceruloplasmin, from PN to Bergmann glia, where ATP7A (Menkes disease protein) is present. The induced cell-specific change in expression restores copper delivery to ceruloplasmin via ATP7A. Overall, the results provide evidence for distinct functions of ATP7A and ATP7B in the cerebellum and illustrate a tight link between copper homeostasis in PN and Bergmann glia.

The distinct functional properties of the nucleotide-binding domain of ATP7B, the human copper-transporting ATPase: analysis of the Wilson disease mutations E1064A, H1069Q, R1151H, and C1104F.

Copper transport by the P(1)-ATPase ATP7B, or Wilson disease protein (WNDP),1 is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P(2)-ATPases are not conserved within the P(1)-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg(2+)-independent manner with a relatively high affinity of 75 microm, compared with millimolar affinities observed for the P(2)-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P(1)-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P(1)-ATPases are distinct from those of the P(2)-ATPases.

Molecular modelling of the nucleotide-binding domain of Wilson's disease protein: location of the ATP-binding site, domain dynamics and potential effects of the major disease mutations.

WNDP (Wilson's disease protein) is a copper-transporting ATPase that plays an essential role in human physiology. Mutations in WNDP result in copper accumulation in tissues and cause a severe hepato-neurological disorder known as Wilson's disease. Several mutations were surmised to affect the nucleotide binding and hydrolysis by WNDP; however, how the nucleotides bind to normal and mutated WNDP remains unknown. To aid such studies, we performed the molecular modelling of the spatial structure and dynamics of the ATP-binding domain of WNDP and its interactions with ATP. The three-dimensional models of this domain in two conformations were built using the X-ray structures of the Ca2+-ATPase in the E1 and E2 states. To study the functional aspects of the models, they were subjected to long-term molecular dynamics simulations in an explicit solvent; similar calculations were performed for the ATP-binding domain of Ca2+-ATPase. In both cases, we found large-scale motions that lead to significant changes of distances between several functionally important residues. The ATP docking revealed two possible modes of ATP binding: via adenosine buried in the cleft near residues H1069, R1151 and D1164, and via phosphate moiety 'anchored' by H-bonds with residues in the vicinity of catalytic D1027. Furthermore, interaction of ATP with both sites occurs if they are spatially close to each other. This may be achieved after relative domain motions of the 'closure' type observed in molecular dynamics simulations. The results provide a framework for analysis of disease mutations and for future mutagenesis studies.

Functional properties of the human copper-transporting ATPase ATP7B (the Wilson's disease protein) and regulation by metallochaperone Atox1.

Wilson's disease protein (WNDP) is a copper-transporting P(1)-type ATPase which plays a key role in normal distribution of copper in a number of tissues, particularly in the liver and the brain. Copper has numerous effects on WNDP, altering its structure, activity, and intracellular localization. To better understand the function of this copper-transporting ATPase and its regulation by copper, we have recently developed the functional expression systems for WNDP and for Atox1, a cytosolic protein that serves as an intracellular donor of copper for WNDP. Here we summarize the results of our experiments on characterization of the enzymatic properties of WNDP and the effects of Atox1 on the WNDP activity.

The role of the invariant His-1069 in folding and function of the Wilson's disease protein, the human copper-transporting ATPase ATP7B.

The copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in ATP7B lead to Wilson's disease, a severe disorder with neurological and hepatic manifestations. One of the most common disease mutations, a H1069Q substitution, causes intracellular mislocalization of ATP7B (the Wilson's disease protein, WNDP). His-1069 is located in the nucleotide-binding domain of WNDP and is conserved in all copper-transporting ATPases from bacteria to mammals; however, the specific role of this His in the structure and function of WNDP remains unclear. We demonstrate that substitution of His-1069 for Gln, Ala, or Cys does not significantly alter the folding of the WNDP nucleotide-binding domain or the proteolytic resistance of the full-length WNDP. In contrast, the function of WNDP is markedly affected by the mutations. The ability to form an acylphosphate intermediate in the presence of ATP is entirely lost in all three mutants, suggesting that His-1069 is important for ATP-dependent phosphorylation. Other steps of the WNDP enzymatic cycle are less dependent on His-1069. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of AMP-PNP is not disrupted by the mutation, the apparent affinity for the nucleotide is decreased by 4-fold. We conclude that His-1069 is responsible for proper orientation of ATP in the catalytic site of WNDP prior to ATP hydrolysis.

Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity.

Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of copper belongs to the recently discovered family of metallochaperones and to copper-transporting P-type ATPases. The mutations in the ATPase ATP7B, the Wilson's disease protein (WNDP), lead to intracellular accumulation of copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the protein-protein interactions between WNDP and the metallochaperone Atox1, suggesting that these interactions are important for normal copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated copper transfer.

Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells.

Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent K(m) of 0.95 +/- 0.25 microm; ADP promotes dephosphorylation with an apparent K(m) of 3.2 +/- 0.7 microm. Replacement of Asp(1027) with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.

Human copper-transporting ATPase ATP7B (the Wilson's disease protein): biochemical properties and regulation.

Wilson's disease protein (WNDP) is a product of a gene ATP7B that is mutated in patients with Wilson's disease, a severe genetic disorder with hepatic and neurological manifestations caused by accumulation of copper in the liver and brain. In a cell, WNDP transports copper across various cell membranes using energy of ATP-hydrolysis. Copper regulates WNDP at several levels, modulating its catalytic activity, posttranslational modification, and intracellular localization. This review summarizes recent studies on enzymatic function and copper-dependent regulation of WNDP. Specifically, we describe the molecular architecture and major biochemical properties of WNDP, discuss advantages of the recently developed functional expression of WNDP in insect cells, and summarize the results of the ligand-binding studies and molecular modeling experiments for the ATP-binding domain of WNDP. In addition, we speculate on how copper binding may regulate the activity and intracellular distribution of WNDP, and what role the human copper chaperone Atox1 may play in these processes.