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J. Biol. Chem., Vol. 277, Issue 49, 46987-46992, December 6, 2002

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Biochemical Characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase^*

Bin Fan and Barry P. Rosen

From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201

Received for publication, August 19, 2002, and in revised form, September 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [-³²P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K_m for ATP of 0.5 mM. CopA has two N-terminal Cys(X)₂Cys sequences, Gly-Leu-Ser-Cys¹⁴-Gly-His-Cys¹⁷, and Gly-Met-Ser-Cys¹¹⁰-Ala-Ser-Cys¹¹³, and a Cys⁴⁷⁹-Pro-Cys⁴⁸¹ motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)₂-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys⁴⁷⁹ or Cys⁴⁸¹ resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 834-residue CopA copper pump from Escherichia coli (1) is a member of the superfamily of cation-transporting P-type ATPases (2). CopA belongs to a subfamily that transports the cations of soft Lewis acids (or, for simplicity, just soft metal cations) (3). Members of one branch of the subfamily transports monovalent cations such as Cu(I) and Ag(I) (4-6). A second branch transports divalent cations such as Zn(II), Cd(II), Pb(II), and Co(II) (7, 8). Copper translocating P-type ATPases are found in virtually every organism, from E. coli to humans and include homologues that are related to the human Menkes (9) and Wilson (10) diseases. In E. coli CopA participates in copper homeostasis and is required for copper efflux and resistance (1).

Distinctive features of the soft metal ion translocating P-type ATPases are one to six N-terminal Cys-(X)₂-Cys sequences that bind soft metal cations in vitro (11-13), and a Cys-Pro-Cys motif in the sixth transmembrane segment (TM)¹ that may be part of the translocation pathway. Thus far intracellular trafficking in eukaryotes is the only physiological role identified for the Cys-(X)₂-Cys sequences (14-17), but this is unlikely to apply to E. coli, which has no intracellular membranes. E. coli CopA contains two N-terminal Cys-(X)₂-Cys sequences, Gly-Leu-Ser-Cys¹⁴-Gly-His-Cys¹⁷ and Gly-Met-Ser-Cys¹¹⁰-Ala-Ser-Cys¹¹³. We have previously shown that none of the four cysteine residues in the two sequences is required for either copper resistance or transport (18). However, a deletion of codons 8-150 (NCopA) lost both copper resistance and transport, suggesting that an N terminus is required even though the cysteines are not.

All P-type ATPases have a conserved aspartate residue that is phosphorylated by ATP during the catalytic cycle. In CopA the corresponding residue is Asp⁵²³. In this report we investigated the ability of CopA to form a phosphorylated intermediate with [-³²P]ATP. The ³²P label on CopA was sensitive to treatment with alkali or hydroxylamine, consistent with acylphosphate formation. Phosphoenzyme formation required a monovalent soft metal cation, either Cu(I) or Ag(I). Divalent Cu(II) or Zn(II) could not substitute. This clearly demonstrates that this copper-translocating P-type ATPase distinguishes between Cu(I) and Cu(II). Paradoxically, mutations of the four cysteine residues in the two N-terminal Cys-(X)₂-Cys sequences increased the apparent affinity for copper, as did deletion of the first Cys-(X)₂-Cys sequence. Mutations in the two cysteine residues in the Cys-Pro-Cys motif in TM6 resulted in loss of copper resistance, transport, and phosphoenzyme formation, indicating that these cysteines play a more critical role in CopA function than the N-terminal cysteines. CopA was solubilized with dodecylmaltoside and purified by metal chelate chromatography. The activity of purified CopA was stimulated by copper or silver ions and inhibited by addition of a Cu(I) chelator.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth of Cells-- Cells of E. coli were grown in Luria-Bertani medium (19) at 37 °C. Ampicillin (100 µg/ml), chloramphenicol (30 µg/ml), isopropyl--D-thiogalactopyranoside (0.1 mM), 5-bromo-4-chloro-3-indolyl--D-galactosidase (80 µg/ml) and L(+)-arabinose (0.0002%) were added as required. To assay inhibition of growth by metal salts, cells were grown overnight in Luria-Bertani medium, diluted 1:100 in the same medium with CuSO₄, and incubated for 6 h at 37 °C with shaking. Growth was monitored from the absorbance at 600 nm. Each strain achieved approximately the same final density in the absence of copper.

Strain Construction and Plasmids-- Standard molecular and genetic techniques were used for strain and plasmid construction (19). The construct of copA deletion strain DC194 and Cys-(X)₂-Cys motif mutants were described previously (18). To introduce mutations in the coding sequence for the cysteines in the Cys-Pro-Cys motif, a sequence corresponding to base pairs 892-1854 of the copA gene was cloned into plasmid pGEM-T Easy (Promega) by polymerase chain reaction (PCR) using forward primer 5'-CTCGGCCATATGCTGGAAGCG-3' and reverse primer 5'-GTAATCGCCGCGGTTTCAATG-3'. Site-directed mutagenesis was carried out using a QuickChange method (Stratagene). The inserts were excised by double digestion with NdeI and SacII and ligated into plasmid pGEXCopA₂ in which copA is inserted into the pGEX-6p-2 vector (Amersham Biosciences). The copA gene was excised from that plasmid by digestion with NcoI and SacII and ligated into pCopA₂ (1). A copA gene with deletion of codons 7-54 (corresponding to amino acid residues 3-18 and including the first Cys-(X)₂-Cys sequence) was generated by PCR amplification of copA of using forward primer 5'-GTTCCATGGCAAAACGCGTGAAAGAAAG-3' and reverse primer 5'-ATGGCCGCCGGCGAAAACCATCACTGC-3', with subsequent cloning into plasmid pGEM-T Easy. The insertion was excised by NcoI/NaeI double digestion and ligation into pCopA₂, generating the plasmid pCopA₂N1. Plasmid pCopA₂N2, which has a deletion of copA codons 7-342 bp (corresponding to amino acid residues 3-114 and includes both Cys-(X)₂-Cys sequences) was generated by PCR using forward primer 5'- GTTCCAATGGCAACCCGCGTACAAAATGCG-3' and the same reverse primer as with pCopA₂N1. The PCR product was excised by NcoI/NaeI double digestion and ligation into plasmid pCopA₂ generating plasmid pCopA₂N2. All mutations were verified by sequencing the entire insert. In pCopA₂ copA is in-frame with the sequence for the Myc epitope and six histidine codons, which are at the 3'-end of copA (1). The wild type and mutant pCopA₂ plasmid were introduced into strain DC194.

Preparation of Everted Membrane Vesicles-- Cells were grown overnight at 37 °C in 5 ml of LB, diluted 100-fold into prewarmed medium and allowed to grow to an optical density of 0.7 at 600 nm. Cells were induced with 0.0002% arabinose for 2.5 h at 30 °C. Everted membrane vesicles were prepared as described previously (1). The membrane vesicles were suspended in a buffer consisting of 25 mM Tris-HCl, pH 7.0, containing 0.25 M sucrose, 0.2 M KCl, and 0.05 mM EDTA and stored at 70 °C until use. Protein concentrations were determined using a bicinchoninic acid method (Sigma Chemical Co.).

Polyacrylamide Gel Electrophoresis and Immunoblotting-- Samples were prepared by incubation in SDS sample buffer for 30 min at room temperature and separated by SDS-PAGE on 8% polyacrylamide gels. Immunoblotting was performed with antibody to the His₆ tag (Clontech) using an enhanced chemiluminescence assay (PerkinElmer Life Sciences) and exposed on x-ray film at room temperature, as described previously (20). To correct for differences in expression of the mutant proteins, the amount of CopA in everted membrane vesicles was normalized to that of the wild type by quantitative Western blot analysis. Each set of membranes was analyzed at four different concentrations, and a standard curve was constructed from the values obtained by densitometry.

⁶⁴Cu Transport Assays-- ⁶⁴Cu was obtained from the Division of Radiological Science, Mallinckrodt Institute of Radiology, Washington University Medical School, which is supported by NCI Research Resource Grant NIH 1 R24 CA86307. Transport assays were performed at room temperature as described previously (1). Unless otherwise noted, the reaction mixture (1 ml) contained 40 mM histidine (pH 6.8), 0.2 M KCl, 0.25 M sucrose, 1 mM dithiothreitol (DTT), 0.5 mg of membrane protein, 10 µM ⁶⁴CuCl₂ (0.5-10 µCi/ml), and 5 mM Na₂ATP. The reaction was initiated by addition of 5 mM MgCl₂. At intervals, 0.1-ml samples were withdrawn and filtered through nitrocellulose filters (0.22-µm pore size, Whatman). The filters were presoaked in a buffer consisting of 40 mM histidine, pH 6.8, 0.2 M KCl, 0.25 M sucrose, 10 mM MgSO₄, and 20 mM CuCl₂. Following filtration, the filters were washed with 5 ml of the same buffer, dried, and the radioactivity quantified by liquid scintillation counting. The values obtained with the assay mixture without membrane vesicles were subtracted from all time points.

Phosphoenzyme Formation with [-³²P]ATP-- Everted membrane vesicles (20 µl of 1 mg/ml protein) were diluted into 140 µl of an assay buffer consisting of 40 mM histidine (pH 6.8), 0.2 M KCl, 0.25 M sucrose, and 5 mM MgCl₂. For assays containing AgNO₃, 0.2 M KNO₃, and 5 mM Mg(NO₃)₂ were used in place of KCl and MgCl₂. Reductant (1.5 µl of 0.1 M DTT, GSH, or cysteine) and 1.5 µl of metal ion solutions or water were added, and the mixture was incubated at room temperature for 5 min. To measure sensitivity to vanadate, 5 mM sodium orthovanadate was added to a final concentration of 50 µM. The mixture was incubated at 4 °C for 5 min, and the reaction was initiated by addition of 2.5 µCi of [-³²P]ATP at a final concentration of 50 µM. After incubation on ice (or at room temperature for purified CopA) for the indicated times, 40 µl of ice-cold 50% tricholoracetic acid was added to terminate the reaction. For pulse-chase analysis, 1 mM cold ATP was added 30 s after the start of the reaction, followed by incubation on ice for another 15 to 30 s. Following addition of tricholoracetic acid, the membrane vesicles were kept on ice for an additional 10 min and then harvested by centrifugation at 15,000 × g for 10 min. The pellet protein was washed once with 0.5 ml of distilled water and once with 0.5 ml of a solution of 50 mM H₃PO₄/NaOH, pH 2.4. To examine alkali lability, the pelleted material was suspended in 0.2 ml of 0.5 M KOH and kept on ice for 5 min. Sensitivity to hydroxylamine was examined by suspending the pelleted material in 0.2 ml of 0.1 M sodium acetate, pH 5.6, followed by the addition of 0.2 ml of 0.25 M NH₂OH, with an additional 10 min incubation at room temperature. The treated samples were again precipitated with tricholoracetic acid and dissolved in 30 µl of 2× concentrated SDS sample buffer diluted with an equal volume of 50 mM H₃PO₄/NaOH, pH 2.4, containing 5% SDS. Samples of 10 µl were loaded on an acidic 8% polyacrylamide gel (21). Following electrophoresis, the gels were stained with Coomassie Blue, dried using a DryEase mini-gel drying system (Novex). Radioactivity was analyzed with a PhosphorImager (Molecular Dynamics).

Purification of CopA-- Membrane vesicles (5 mg/ml) were solubilized in buffer D, consisting of 25 mM Tris-HCl, pH 7.0, 50 mM KCl, 1 µM CuCl₂, 5 mM MgCl₂, and 10% glycerol, containing 2% dodecyl maltoside (DDM). The mixture was gently shaken at 4 °C for 1 h, and the insoluble fraction was removed by centrifugation at 250,000 × g for 1 h. The soluble fraction was loaded onto a Probond Ni²⁺ affinity column (Invitrogen) pre-equilibrated with buffer D containing 0.1% DDM. The column was washed with 5 bed volumes of the same buffer, followed by 5 bed volumes of the same buffer containing 40 mM imidazole. The protein was eluted with the same buffer containing 0.1 M imidazole. Fractions of 0.5 ml were collected into tubes containing concentrated DTT and EDTA such that the final concentrations became 1 and 0.1 mM, respectively. The samples were analyzed for CopA by SDS-PAGE and immunoblotting. CopA-containing fractions were concentrated 10-fold using Centricon concentrators (Millipore), and the imidazole was removed by gel filtration on a 5-cm column filled with 3 ml of Sephadex G-25 column pre-equilibrated with buffer D containing 0.1% DDM. All buffers were degassed under vacuum or bubbled with Argon before use. All steps were performed at 4 °C.

ATPase Assays-- ATPase activity was estimated by a coupled spectrometric assay (22). The reaction mixture (0.4 ml) contained 40 mM histidine, pH 6.8, 50 mM KCl, 10% glycerol, 0.1% DDM, 0.4 mg of total E. coli lipids (AVANTI polar-lipids), 0.25 mM NADH, 1.25 mM phosphoenolpyruvate, 7 units of pyruvate kinase, 10 units of lactate dehydrogenase, 5 mM ATP. Where indicated 1 mM NaN₃, 0.25 mM BCS, 10 µM CuCl₂ or 10 µM Cu(I) (acetonitrile copper(I) hexafluorophosphate, Sigma) were added. The reaction mixture was incubated at 37 °C for 5 min prior to initiating the assay with 5 mM MgCl₂.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cu(I)-dependent Formation of an Acylphosphate Bond in CopA-- A signature property of the P-type ATPases is that the conserved aspartate residue in the DKTG motif accepts the -phosphate from ATP during the catalytic cycle, forming a covalent acylphosphate intermediate (23). To investigate phosphoenzyme formation in CopA, everted membrane vesicles were prepared from cells of strain DC194 (copA) expressing wild type copA on a plasmid. CopA was phosphorylated with [-³²P]ATP in a time-dependent manner, with maximum labeling within 30 s (Fig. 1A). Vesicles from the copA deletion strain DC194 did not exhibit a radioactive band at the corresponding position (Fig. 1, lane 1). The intermediate was sensitive to basic pH and hydroxylamine (Fig. 1B), which are considered to be reliable criteria for the presence of an acylphosphate bond. The label could be chased by 1 mM unlabeled ATP within 15 s, indicating the transient nature of the intermediate. In the presence of 50 µM sodium orthovanadate, which mimics the transition state when bound in the active site of P-type ATPases, the reaction was entirely inhibited. On the other hand, phosphoenzyme formation was not inhibited by azide, an inhibitor of many ATPases.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Properties of CopA acylphosphate intermediate formation. Membrane vesicles were prepared from cells of E. coli strain DC194 (copA) bearing plasmid pCopA2 or derivatives with copA mutations. The reaction was initiated by addition of 50 µM [-32P]ATP (~2.5 µCi) at 4 °C. Samples were analyzed on 8% polyacrylamide gels, as described under "Experimental Procedures." Radioactivity was imaged and quantified with a phosphorimager. A, time dependence of acylphosphate intermediate formation. Each reaction contained 20 µg of membrane protein from cells expressing wild type CopA and 10 µM CuCl2 with or without 1 mM DTT, as indicated. Reactions were terminated at the indicated times by additional of trichloroacetic acid. B, properties of acylphosphate formation in wild type CopA and quadruple cysteine mutant. Wild type and C14A/C17A/C110A/C113A CopAs were reacted with [-32P]ATP for 30 s, following which the label was chased with 1 mM nonradioactive ATP for an additional 15 or 30 s. In the other reactions, membranes were incubated with the following inhibitors for 5 min prior to addition of [-32P]ATP: 50 µM orthovanadate, 0.25 M hydroxylamine, 0.5 M KOH or 1 mM NaN3. C, effect of reductants and metals. Membrane vesicles containing wild type CopA were incubated for 5 min with the indicated additions prior to addition of [-32P]ATP for 30 s: 1 mM DTT, 1 mM GSH, 1 mM cysteine, 10 µM CuCl2, 0.2 mM ZnSO4, 0.2 mM CoSO4, 10 or 20 µM AgNO3, 10 µM acetonitrile copper(I) hexafluorophosphate, or 50 µM BCDS

Since CopA is a copper ion pump, acylphosphate formation would be expected to be stimulated by copper ion. In the absence of copper ion, no radioactive band was observed (Fig. 1C, lane 1). To date there has been no clear determination of the redox state of the substrate of copper ion pumps, that is, whether these ATPases transport Cu(II), Cu(I) or both. Our previous work showed that a reductant, DTT, was required for the transport of ⁶⁴Cu with membrane vesicles (1, 18). To investigate whether reductant is also required for phosphoenzyme formation, several reductants were added to the assay. In agreement with the results of the transport assays, a significant radioactive band was observed only in the presence of Cu(II) and reductant: DTT, GSH, or cysteine (Fig. 1C). DTT and GSH were more effective than cysteine. For those reasons DTT was usually added as reductant in subsequent assays.

The requirement for reductant could be to reduce Cu(II) to Cu(I) or to reduce the cysteines in CopA or both. For that reason the effect of mono- and divalent soft metal ions on acylphosphate formation was examined (Fig. 1C). No phosphoenzyme formation was observed in the presence of the divalent cations Zn(II) or Co(II), whether or not DTT was present. In contrast, either Ag(I) or Cu(I) (added in the form of acetonitrile copper(I) hexafluorophosphate) stimulated phosphoenzyme formation even in the absence of DTT, showing that DTT is not necessary to maintain the enzyme in a reduced state. Moreover, bathocuproindisulfonate (BCDS), which chelates Cu(I) and not Cu(II), prevented labeling. These results demonstrate that CopA is a monovalent (but not a divalent) soft metal ion P-type ATPase.

Requirement of the N Terminus for CopA Activity-- One characteristic that differentiates soft metal P-type ATPases from their hard metal homologues is the presence of one or more Cys-(X)₂-Cys metal binding domains in the cytosolic N-terminal region. We have previously shown that none of the four cysteine residues in the Gly-Leu-Ser-Cys¹⁴-Gly-His-Cys¹⁷ and Gly-Met-Ser-Cys¹¹⁰-Ala-Ser-Cys¹¹³ sequences of CopA is required for either copper resistance or transport, although deletion of codons 8-150 (NCopA) lost both copper resistance and transport (18). To extend this observation, two N-terminally truncated CopAs were constructed: pCopA₂N1, which is truncated immediately after the first Cys-(X)₂-Cys motif, and pCopA₂N2, which has both Cys-(X)₂-Cys motifs deleted. Cells expressing N1 CopA retained partial resistance to CuSO₄ in vivo, while cells expressing N2 CopA were as sensitive as the CopA deletion strain (Fig. 2), even though the truncated proteins were made in amounts comparable to that of the wild type (Fig. 3A). These results suggest that at least a portion of the cytosolic N terminus of the protein is necessary, although whether that is for activity or proper folding cannot be deduced from these results.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of N-terminal truncations on copper resistance. Copper ion resistance was assayed in strain DC194 (copA) () or DC194 bearing plasmid pCopA2 (wild type) (), plasmid N1pCopA2 (deletion of CopA residues 7-54) (), or plasmid N2pCopA2 (deletion of CopA residues 3-113) ().

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Acylphosphate intermediate formation in CopA mutants. Phosphorylation of CopA was assayed as described in the legend to Fig. 1. Each lane contained 20 µg of membrane protein from cells of DC194 without a plasmid (lane 1) or bearing plasmids expressing wild type CopA (lane 2), C14A/C17A (lane 3), C110A/C113A (lane 4), C14A/C17A/C110A/C113A (lane 5), N1 (lane 6), N2 (lane 7), C479A (lane 8), C481A (lane 9), or C481H (lane 10). A, immunoblotting with antibody against the His6 tag. B, labeling of CopA with [-32P]ATP, with radioactivity in each lane imaged with a phosphorimager.

CopA mutants with alanine substitutions in the first (C14A/C17A) or second (C110A/113A) Cys-(X)₂-Cys motifs or with all four cysteines substituted (C14A/C17A/C110A/C113A) formed an acylphosphate intermediate equivalent to wild type CopA (Figs. 1B and 3B). Indeed, deletion of the N terminus through the first Cys-(X)₂-Cys motif did not appear to affect the ability of the N1 CopA to form a phosphorylated intermediate, just as a strain expressing this mutant copA conferred copper resistance in vivo. Consistent with the loss of resistance in cells bearing pCopA₂N2, the N2 enzyme did not form a phosphorylated intermediate.

Requirement of an Intramembrane CPC Motif for CopA Activity-- A distinctive feature of soft metal ATPases is a highly conserved Cys-Pro-Cys motif in TM6, the putative translocation domain (24). The Cys-Pro-Cys motif in CopA includes Cys⁴⁷⁹ and Cys⁴⁸¹. The two cysteines were individually mutated to C479A and C481A. Since the Enterococcus hirae CopB has a Cys-Pro-His sequence (6), a C481H mutant was also constructed. All three mutant proteins were expressed in amounts comparable to wild type CopA (Fig. 3A). The effect of the mutations on copper resistance in vivo (Fig. 4A) and ATP-driven ⁶⁴Cu transport in vitro were examined. None of the mutant genes conferred resistance to copper, and everted membrane vesicles from cells expressing those genes were unable to accumulate ⁶⁴Cu (Fig. 4). No phosphoenzyme intermediates were detected in the C479A, C481A, or C481H proteins (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Copper resistance and transport by CopA Cys-Pro-Cys mutants. A, copper ion resistance was assayed in strain LMG194 (wild type) (), DC194 (copA) (), or DC194 bearing a plasmid with the gene for wild type CopA (), C479A (), C481A (), or C481H (). B, uptake of 64Cu in everted membrane vesicles of E. coli strain DC194 () or DC194 expressing wild type CopA (), C479A (), C481A (), or C481H (). Vesicles were prepared as described under "Experimental Procedures." Cells were induced with 0.0002% arabinose as described under "Experimental Procedures." Transport was assayed with 10 µM 64CuCl2 reduced with 1 mM DTT.

Copper Dependence of Phosphoenzyme Formation-- Formation of the acylphosphate intermediate with increasing copper concentration was investigated (Fig. 5). Wild type CopA, the C14A/C17A/C110A/C113A quadruple mutant, and the N1 mutant each responded to increasing concentrations of copper (Fig. 5A). The bands from three separate ³²P-labeled gels were quantified by densitometry. The values were averaged and, for clarity, were normalized to the value at 50 µM (Fig. 5B). The apparent K_m for copper of wild type CopA was 1.5 ± 0.5 µM, which is similar to the K_m of 3.9 µM for the copper dependence of ATPase activity of the Achaeoglobius fulgidus CopA (25). The apparent K_m of the quadruple mutant was 0.45 ± 0.1 µM, and the N1 exhibited an apparent K_m of 0.84 ± 0.2 µM. Thus, removal of the first N-terminal Cys-(X)₂-Cys motif or modification of all four N-terminal cysteines seemed to produce an apparent increase in affinity for Cu(II).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Copper dependence of phosphoenzyme formation. Phosphorylation of CopA was assayed as described in the legend to Fig. 1. Radioactivity was imaged and quantified with a phosphorimager. A, each lane of an 8% SDS gel contained 20 µg of membrane protein labeled with [-32P]ATP from cells of DC194-bearing plasmids expressing wild type CopA (row 1), C14A/C17A/C110A/C113A (row 2), or N1 (row 3) at the indicated concentrations of CuCl2 without or with 1 mM DTT. B, phosphoenzyme formation was quantified by densitometry. Values were normalized to the level of phosphorylation in the presence of 50 µM Cu(II). The lines represent best fits of the data to the Michaelis-Menten equation using SigmaPlot and generated half maximal stimulatory concentrations of copper of 1.5 ± 0.5 µM (squares, wild type), 0.45 ± 0.1 µM (inverted triangles, quadruple cysteine mutant), and 0.84 ± 0.2 µM (circles, N1)

Solubilization and Purification of CopA-- Membrane vesicles were prepared from arabinose-induced cells bearing plasmid pCopA2, which carries a copA gene in-frame with the sequence for the Myc epitope and six histidine codons under control of the arabinose promoter. The vesicles were solubilized with 2% dodecylmaltoside, and CopA was purified by metal chelate affinity chromatography (Fig. 6A). The presence of a small amount of copper during solubilization and purification was found to result in more active and stable CopA. A minor band was observed upon immunoblotting (Fig. 6B). Since the slightly smaller protein reacted with antibody to the histidine tag, there was probably some degradation at the N terminus of CopA. Although the purified protein exhibited ATPase activity, hydrolysis was inhibited 80-90% by sodium azide. Since phosphoenzyme formation is not inhibited by sodium azide (Fig. 1B), it is likely that the azide-sensitive ATPase activity is due to minor contamination by a highly active ATPase.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Purification and properties of CopA. Membrane vesicles from strain DC194 bearing plasmid pCopA2 were solubilized with 2% DDM and purified on a Probond Ni2+ column, as described under "Experimental Procedures." Samples were analyzed by SDS-PAGE on two 8% polyacrylamide gels. A, gel was stained with Coomassie Blue. Lane 1, 40 µg of membrane protein from strain DC194 (copA); lane 2, 40 µg of membrane protein membranes from strain DC194 pCopA2; lane 3, 40 µg of protein from the DDM extract of membranes from strain DC194 pCopA2; lane 4, flow-through from Probond column; lane 5, eluate with 40 mM imidazole; lane 6, eluate with 100 mM imidazole; lane 7, 5 µg of pooled and concentrated CopA-containing fractions. The volume of sample in lanes 4-6 was adjusted to add an amount equivalent to that in lane 3. B, immunoblot with antibody to the His6 tag. Each lane of gel B was the same as gel A with one-tenth the amount of protein. The migration of standard proteins is indicated by the left arrows. C, kinetics of ATPase activity of purified CopA. The ATPase activity of purified ATPase was determined in the presence of 10 µM CuCl2, 1 mM DTT, and the indicated concentrations of ATP. Each value is the average of two separate assays. The data fitted to the Michaelis-Menten equation using SigmaPlot yielded a Km of 0.52 ± 0.05 mM and Vmax of 0.19 ± 0.06 µmol/min/mg protein.

The azide-insensitive ATPase activity of 130 nmol/mg/min was inhibited to 65 nmol/mg/min by BCS, a Cu(I) chelator, that may reflect residual copper remaining from purification. Neither Cu(II) nor Zn(II) stimulated ATPase activity. Compared with the rate in the presence of BCS, there was an ~4-fold activation by Cu(I) (280 nmol/mg/min) or Cu(II) plus DTT (295 nmol/mg/min). In other experiments, Ag(I)-stimulated activity was 4- to 5-fold higher than the rate with BCS. The Ag(I)-stimulated activity exhibited a relatively broad pH profile, with approximately equal activity at pH 7 and 8, and about one-third lower at pH 6. These results indicate that the azide-insensitive ATPase activity is catalyzed by CopA in a reaction that requires either Cu(I) or Ag(I). Cu(I)-stimulated ATPase exhibited an apparent K_m for ATP of 0.5 mM (Fig. 6C). The Cu(I)-stimulated V_max of 0.19 µmol/mg/min is similar to the V_max for the Ag(I)-stimulated ATPase reported for the CopA homologue from A. fulgidus (25).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The distinctive feature of P-type ATPases that gave the superfamily its name is the formation of an acylphosphate on a conserved aspartate residues during the catalytic cycle (23). Phosphoenzyme formation has been shown for several copper-translocating P-type ATPase homologues of CopA (25-28). In this report we demonstrate Cu(I)-dependent phosphorylation of CopA. Phosphorylation was sensitive to alkali and hydroxylamine, consistent with acylphosphate formation on the conserved Asp⁵²³.

Whereas it is presumed that copper pumps are specific for Cu(I) and not Cu(II), this has not been proven. Since an N-terminal peptide containing the Cys-(X)₂-Cys motifs of the Wilson disease protein has been shown to bind Cu(II) and other divalent cations (29), it is possible that these proteins could pump both Cu(I) and Cu(II). Phosphorylation of WND and E. hirae CopA were not copper-dependent. This could have been due to copper contamination in the buffers, since copper chelators were found to inhibit. Phosphorylation of MNK and the A. fulgidus CopA showed copper dependence, but it was not clear whether the activity was stimulated by Cu(I), Cu(II), or both. In both cases DTT was present in the phosphorylation assays, which would reduce some or all of the Cu(II) to Cu(I), but DTT might also have been necessary to reduce cysteine thiolates in the proteins. The data reported here clearly show that Cu(I) and not Cu(II) stimulates phosphorylation of CopA, and that DTT is not required. While phosphoenzyme formation was stimulated by Cu(II) in the presence of DTT, Cu(I) and Ag(I) were equally effective in the absence of DTT. Thus the requirement for DTT appears to be in reduction of copper rather than for maintenance of protein cysteine thiolates. There was no phosphorylation in the presence of Cu(II) or other divalent cations, so CopA is not a divalent cation pump.

The soft metal P-type ATPase are distinguished from their hard metal homologues by the presence of N-terminal metal binding motifs, usually Cys-(X)₂-Cys, and a second cysteine motif, usually Cys-Pro-Cys, in TM6. The physiological role of these motifs and the function of the individual cysteine residues in the biochemical mechanism of the ATPases are open questions. In eukaryotes the N-terminal motifs may interact with metal chaperones or may be involved in copper-regulated trafficking of copper pumps from intracellular compartments to the plasma membrane. Most prokaryotes lack intracellular membranes or compartments, and the function of the Cys-(X)₂-Cys motifs in their copper pumps is less clear. In E. coli there are no intracellular membranes, and no metallochaperones have been identified. In this report we investigated the ability of N-terminal cysteine mutants and deletions to form a phosphorylated intermediate. A quadruple mutant lacking all four cysteine residues of the two N-terminal Cys-(X)₂-Cys motifs was phosphorylated by [-³²P]ATP. CopA with a deletion of the first Cys(X)₂Cys motif similarly was phosphorylated. In both there was a 2-3-fold decrease in the concentration of copper required for maximal phosphoenzyme formation. Does this apparently counterintuitive result imply that binding of copper to the N-terminal motifs decrease the affinity of the pump for copper, perhaps for a regulatory function? An alternative explanation is that in vitro the Cys-(X)₂-Cys motifs reduce access of copper to the translocation pathway or reduce the local concentration of copper in that region. The mutations did not produce altered copper resistance, so it is questionable whether the in vitro results reflect an increased efficiency of the pump in vivo. While none of the four N-terminal cysteines are required for CopA activity under the conditions studied, their contributions to CopA function may be apparent only under different physiological conditions. Similar conclusions have been made about the Cys-(X)₂-Cys motifs in other copper P-type ATPases. For example, in MNK mutation or deletion of the first four of the six Cys-(X)₂-Cys motifs had no apparent effect on copper-induced trafficking, their only identified function (15). Although none of the cysteine residues of the two Cys-(X)₂-Cys motifs of CopA are obligatory, deletion of the N terminus to just before the first putative transmembrane region resulted in loss of function, but it is possible that the remainder of the protein does not fold properly.

In three CopA homologues the Cys-Pro-Cys motif that is distinctive in soft metal ion-translocating ATPases has been shown to be located in TM6 (30-32), so it is reasonable to assume that it is in TM6 of CopA as well. The Cys-Pro-Cys motif has been proposed to be part of the translocation domain (24). To examine the requirement of the cysteine residues, Cys⁴⁷⁹ and Cys⁴⁸¹ were changed to alanine residues. Cells expressing either mutant became sensitive to copper. Both mutant proteins were found in the membrane in normal amounts, but neither was phosphorylated by [-³²P]ATP. Since the CopB homologue from E. hirae has a Cys-Pro-His motif in the corresponding TM6 (6), we constructed a C481H mutant. Cells expressing the mutant CopA were also sensitive to copper, and the protein with a Cys-Pro-His motif did not form an acylphosphate intermediate. A reasonable interpretation of these results is that the Cys-Pro-Cys motif of CopA is essential for Cu(I) binding, and that mutants with Cys-Pro-His, Cys-Pro-Ala, or Ala-Pro-Cys no longer bind metal. This is consistent with the known catalytic cycle for hard metal ion-translocating P-type ATPases, where binding of metal is required for acylphosphate formation at the conserved aspartate residue (33). Thus, we propose that the Cys-Pro-Cys residues of CopA are required for binding of Cu(I) in the ion translocation pathway and hence for Cu(I) transport.

	FOOTNOTES

^* This research was supported by National Institute of General Medical Sciences Grant GM52216 and American Heart Association Predoctoral Fellowship 0215161Z (to B. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1512; Fax: 313-577-2765; E-mail: brosen@med.wayne.edu.

Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M208490200

	ABBREVIATIONS

The abbreviations used are: TM, transmembrane segment; DTT, dithiothreitol; BCDS, bathocuproindisulfonate; DDM, dodecyl maltoside, MNK, Menkes disease-related protein; WND, Wilson disease-related protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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