Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance.

Intracellular copper routing in Enterococcus hirae can be accomplished by the CopZ metallochaperone. Using surface plasmon resonance analysis, we show here that CopZ interacts with the CopA copper ATPase. The binding affinity of CopZ for CopA was increased in the presence of copper, due to a 15-fold lower dissociation rate constant. Mutating the N-terminal copper binding motif of CopA from CxxC to SxxS abolished this copper-induced effect. Moreover, CopZ failed to show an interaction with an unrelated copper binding protein used as a control. These results show that (i) the CopA copper ATPase specifically interacts with the CopZ chaperone, (ii) this interaction is based on protein-protein interaction, and (iii) surface plasmon resonance is a novel tool for quantitative analysis of metallochaperone-target interactions.

Copper in disorders with neurological symptoms: Alzheimer's, Menkes, and Wilson diseases.

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-beta-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimer's disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimer's disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimer's disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites.

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.

The Menkes copper transporter is required for the activation of tyrosinase.

Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux. A role for MNK at the TGN in mammalian cells has not been demonstrated. In this study, we investigated whether the MNK protein is required for the activity of tyrosinase, a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. We demonstrate that recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express MNK protein there was substantial tyrosinase activity. Co-expression of the Menkes protein and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of MNK. Collectively, these findings suggest that the MNK protein transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes and reveal a second copper transport role for MNK in mammalian cells. These findings describe a single cell-based system that allows both the copper transport and trafficking functions of MNK to be studied. This study also contributes to our understanding of the molecular basis of pigmentation in mammalian cells.

Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K(m) and V(max) values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein.

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.

CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper-inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to -11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA-repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY-DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10(-9) M and 5 x 10(-9) M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 microM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.

Functional expression of the Enterococcus hirae NaH-antiporter in Escherichia coli.

We recently described the cloning of napA, the putative structural gene for the NaH-antiporter of Enterococcus hirae (Waser, M., Bienz-Hess, D., Davies, K., and Solioz, M. (1992) J. Biol. Chem. 267, 5396-5400). To analyze the gene product of napA, we expressed it in Escherichia coli. When placed under the control of a T7 promoter, napA could be transcribed and labeled specifically with [35S]methionine. The resultant gene product exhibited an apparent M(r) of 3,4 x 10(4) when subjected to sodium dodecyl sulfate-gel electrophoresis. The function of NapA was tested by expressing it from its own promoter in the E. coli mutant EP432. This mutant lacks both of the endemic NaH-antiporters, NhaA and NhaB; its growth is thus very sensitive to Na+ and Li+ and membranes derived from this strain do not exhibit NaH-antiport activity. When complemented with napA, EP432 gained tolerance to Na+ or Li+. Membranes prepared from the complemented mutant exhibited NaH-antiport activity. The properties of this activity were determined by acridine fluorescence measurements on vesicles energized with lactate. The NaH-antiporter expressed by napA exhibited a Km of 1 mM for Na+ and 0.1 mM for Li+ at pH 7.5. At pH 8.5, the relative rate of NaH-antiport activity was 50%, with little change in the Km, and approached zero at pH 9. These results demonstrate that napA is the structural gene for the NaH-antiporter of E. hirae. NapA exhibits properties different from those of the two E. coli NaH-antiporters encoded by nhaA and nhaB, yet functionally complements a defect in these genes.