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J. Biol. Chem., Vol. 275, Issue 25, 18611-18614, June 23, 2000

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ACCELERATED PUBLICATION
Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper Transporter, Ccc2^*

David L. Huffman^§ and Thomas V. O'Halloran^¶

From the  Department of Chemistry and the ^¶ Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3113

Received for publication, March 16, 2000, and in revised form, April 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Copper is an essential cofactor in hydrolytic, electron transfer, and oxygen utilization enzymes and is also crucial for high affinity iron uptake in yeast (1, 2). Copper uptake in eukaryotes is mediated by the CTR plasma membrane proteins (3-6). Once inside the cell, a portion of the copper is delivered to P-type ATPases, which pump this metal ion into vesicles for ultimate incorporation into multicopper oxidases. In yeast the P-type ATPase is Ccc2 (7-9) and the multicopper oxidase is Fet3 (10). Until recently, copper was thought to be delivered to target proteins as glutathione complexes; however, the Atx1 metallochaperone protein, a cytosolic Cu(I) receptor, is required downstream of CTR1 and upstream of Ccc2 and related ATPases (11, 12).

Copper chaperone proteins are soluble, intracellular receptors that bind and deliver copper to specific partner proteins (12). Initially identified as antioxidant and biosynthetic pathway proteins, the yeast metallochaperone Atx1 and the copper chaperone for superoxide dismutase (CCS)¹ have since been shown to be Cu(I) receptors that interact with specific vesicular and cytoplasmic targets, respectively (12-15).

Copper-trafficking pathway proteins including homologues of CTR1 (5), Atx1 (16), Cox17 (17), Ccc2 (18-22), and Fet3 (2, 23) are highly conserved from yeast to humans. Complementation studies demonstrate that human Atx1 (HAH1) functions in place of yeast Atx1 (24), and the Wilson's and Menkes' disease proteins, homologues of Ccc2, function in place of Ccc2 to deliver copper to Fet3 (25-27).

Recent structural studies provide insights into the intracellular chemistry of copper transfer (28). Crystallographic studies of Atx1 indicate that the metal-binding motif is housed in a surface-exposed loop, with the cysteines located in the first loop and the first -helix (29). This fold is conserved in other eukaryotic copper-trafficking proteins including Menkes' ATPase domain 4, MNK4 (30), and the CCS metallochaperone domain I (31). The two N-terminal domains of Ccc2 are predicted to possess this same -fold, albeit with multiple negatively charged surface residues like MNK4, its human counterpart. In contrast, Atx1 possesses multiple positively charged lysines on its surface (29). Mutation of conserved lysines on the surface of Atx1 greatly reduces the copper-dependent interaction of Atx1 and Ccc2 in vivo (12, 32).

Cytoplasmic free Cu(I) is notably unavailable to the high affinity copper enzymes such as SOD1 (superoxide dismutase) (14). In fact, the results are consistent with the presence of a vast excess of specific and nonspecific copper chelation sites relative to the total number of copper atoms per cell. Furthermore, copper exchange rates are generally thought to be slow because of the strength of the Cu(I) ligand bonds (33). These considerations raise the dilemma of how a copper trafficking protein can bind its cargo tightly enough to prevent loss to inappropriate binding sites and yet allow facile release at specific destinations. To address these issues, the energetics of copper transfer between Atx1 and Ccc2 were probed using a direct assay of metal occupancy.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Isolation of Ccc2a and Atx1-- The gene encoding the 72-residue N-terminal Atx1-like domain of Ccc2, Ccc2a, was cloned into pET11d (Novagen) and resequenced. The resultant plasmid, pDLHV021, was transformed into Escherichia coli strain BL21(DE3) and induced with 1 mM isopropyl-1-thio--D-galactopyranoside in M9 supplemented with cas-amino acids. The protein was extracted from the cell pellet by freeze-thaw extraction in 20 mM MES/Na, 1 mM EDTA, 10 mM DTT. After batch treatment with CM-Sepharose (Amersham Pharmacia Biotech), filtrate was loaded onto a DEAE-Sepharose CL-6B (Amersham Pharmacia Biotech) column, pre-equilibrated with 20 mM MES/Na, 0.1 mM EDTA, 10 mM DTT, pH 6.0 and eluted with a linear NaCl gradient. Ccc2a-containing fractions were combined, concentrated, and loaded onto a Superdex 75 (Amersham Pharmacia Biotech) column and eluted with 20 mM MES/Na, 150 mM NaCl, 10 mM DTT. Approximately 3 mg of pure protein was obtained per liter of culture. Electrospray mass spectroscopy revealed a mass of 7882.1 daltons, consistent with the processing of the N-terminal Met in the expression strain. The cloning procedure introduced an Ala after the N-terminal Met; therefore, the recombinant protein begins with Ala at position 1 and ends with Ser at position 72.

The concentration of a Ccc2a stock solution was determined by hydrolysis (Harvard Microchem) giving  = 2750 M¹ cm¹ (at 280 nm) in 20 mM MES/Na, pH 6. The hydrolysis results indicate that the Bradford method (34) using IgG as a standard underestimates the concentration of Ccc2 that can be accounted for if multiplication factor of 2.9 is applied to the IgG-based value. Atx1 was purified as described previously (12), and the concentration was determined as described for Ccc2a. Total amino acid hydrolysis (Harvard Microchem,  = 4950 M¹ cm¹ in 50 mM Tris/MES, pH 8) data indicate that the Bradford assay (34) using IgG standards leads to an overestimate of the concentration of Atx1. In this case, a multiplication factor of 0.54 was applied to the IgG standard curve.

Preparation of Protein Samples for Metal Transfer-- Frozen protein stocks were treated with excess DTT, washed, and exchanged into the metal insertion buffer immediately prior to loading with copper. Cu(I)-Atx1 and Cu(I)-Ccc2a were prepared in an N₂ atmosphere chamber (Vacuum Atmospheres) at 12 °C by adding 1 equivalent of [Cu(I)-(CH₃CN)₄]PF₆ in CH₃CN to a solution of apoprotein in 50 mM Tris/MES, pH 8, with stirring. Unbound metal was removed by washing with at least 3 volumes of buffer in an ultrafiltration device.

The metal to protein stoichiometries for Atx1 and Ccc2a are typically ~1, even when the proteins are incubated with excess Cu(I) in the presence of glutathione (GSH) or DTT. The protein samples used for metal transfer were: Cu(I)-Atx1, 760 µM, Cu/protein ratio 1.0; Cu(I)-Atx1, 2000 µM, Cu/protein ratio 0.94; Cu(I)-Ccc2a, 1890 µM, Cu/protein ratio 0.62; apoAtx1, 770 µM; apoCcc2a, 520 µM; apoCcc2a, 2030 µM. Metal concentration was determined by ICP-AES. All samples were stored at 4 °C or 20 °C in an N₂ atmosphere prior to use.

Metal Transfer Assay-- Metal ion transfer experiments were performed by combining Cu(I)-Atx1 or Cu-Ccc2a with apoCcc2a or apoAtx1 in an inert atmosphere maintained at 18 °C at pH 6 in 20 mM MES/Na or at pH 7.2 in 4 mM NaP_i. After incubation, the mixture was separated using the same buffer (pH 6 or pH 7.2 as indicated) with a linear gradient (0-0.5 M NaCl) on a Bio-Scale Q2 (Biologic) or a RESOURCE Q (1 ml, Amersham Pharmacia Biotech). Column fractions were analyzed for protein and metal content by the Bradford assay (34) and ICP-AES, respectively. Control experiments indicate routine protein recovery rates of more than 95%.

Determination of Equilibrium Constant-- The equilibrium expressions for Cu(I) exchange are shown in Equations 1 and 2.

(Eq. 1)

(Eq. 2)

Using expressions for [Atx1]_eq and [Ccc2a]_eq,

(Eq. 3)

(Eq. 4)

(Eq. 5)

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This in vitro assay of Cu(I) transfer from the metallochaperone Atx1 to its physiological partner, Ccc2, provides a direct test of copper trafficking function. When CuAtx1 is incubated with apoCcc2a, the metal partitions between the two proteins (Fig. 1). Control experiments demonstrate that Atx1 is not retained on the Q2 strong anion column (Fig. 1b), whereas Ccc2a binds strongly.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Copper transfer assay between CuAtx1 and Ccc2a. Protein (,) and metal ion (,- - -) concentrations of fractions from strong anion exchange chromatography. Controls demonstrate that Ccc2a strongly binds and elutes from the column in fractions 24-26 (20 mM MES/Na, pH 6, ~0.35 M NaCl) (a), whereas CuAtx1 does not bind to the column and elutes in fractions 5-7 (20 mM MES/Na, pH 6) (b). c, metal ion transfer assay between CuAtx1 and Ccc2a demonstrates that Cu(I) is transferred to Ccc2a.

These experiments also provide a quantitative insight into the thermodynamics and kinetics of metal transfer. To test the equilibrium assumption, the reverse reaction of CuCcc2a with apoAtx1 was conducted. As shown in Table I, CuCcc2a can transfer metal to apoAtx1. The reverse reaction gives the same product distribution as the forward reaction, establishing that the system reaches equilibrium in the time frame of the experiments. Furthermore, the product distribution was independent of the incubation time, indicating that copper exchange is complete even at the shortest possible incubation times (1 min).

The copper exchange constant (K_exchange) was obtained from a linear least squares fit of the data in Table I to the modified form of the equilibrium expression shown in Equation 5. Total moles of loaded and recovered copper were routinely found in the 90%+ range, and runs with less than 86% recovery were not used. The linear least squares fit of the data (corresponding to experiments 1-14) over a range of protein concentrations supports the equilibrium assumption implicit in Equation 5 (Fig. 2), revealing a slope (K_exchange) = 1.4 and standard deviation s_m = 0.1 (correlation coefficient, r, of 0.95). Data for both the forward and reverse reactions fit equally well, further indicating that the system is at equilibrium. The averages of the K_exchange values at pH 6.0, 1.5( = 0.2), and at pH 7.2, 1.2( = 0.3), are the same within experimental error indicating no effect of pH in this range (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Determination of exchange constant, Kexchange. The plot of Equation 5 is shown with experiments 1-14 from Table I. A linear regression analysis yields a slope = 1.4 (corresponding to the equilibrium constant, Kexchange) with a standard deviation sm = 0.1 and a correlation coefficient r = 0.95.

To determine whether copper competitors can interfere with the transfer or remove the copper ion from these proteins, we conducted the metal transfer experiment in the presence of a physiologically relevant Cu(I) binding agent, GSH. GSH avidly binds Cu(I) to form Cu(GSH)₂ with a stability constant of ₂ = 10^38.8 (35). The addition of physiological levels of GSH (1-5 mM) does not effect the final equilibrium position (Table I, experiments 15-17). The copper transfer reaction is unaffected by the presence of even a 50-fold molar excess of GSH over the Cu(I) concentration (experiment 17, Table I), indicating that low molecular weight thiols do not participate in this equilibrium. We also tested whether Cu-Atx1 could transfer copper to another copper-binding protein by incubating 190 µM CuAtx1 anaerobically with 167 µM bovine serum albumin at pH 6 for 5 min. No copper is transferred from Cu-Atx1 to bovine serum albumin in four separate determinations. Thus, the strong Cu(I) thiolate bonds in Atx1 not only protect Cu(I) from disproportionation and air oxidation (12) but may also prevent loss of the metal ion to adventitious binding sites within the cell.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When the copper-bound form of the chaperone is directly incubated with apoCcc2a, Cu(I) equilibrates between the two proteins, and it does so rapidly. A stepwise mechanism for direct and rapid metal transfer is proposed. In the first step, CuAtx1 docks apoCcc2a. A specific orientation between CuAtx1 and apoCcc2a could poise the Cu(I) center for nucleophilic attack by thiol from the adjacent protein, forming a Cu(S-Cys)₃ intermediate. In the next step, the copper rapidly partitions between the two metal-binding sites within the protein-protein complex via the formation and decay of two- and three-coordinate copper thiolate intermediates. In the final step, the complex dissociates to provide apoAtx1 and Cu-Ccc2a.

Thermodynamic versus Kinetic Control of Copper Trafficking-- The metal exchange results shown herein demonstrate that the thermodynamic gradient for copper transfer between this copper chaperone and its target domains is ~0.2 kcal/mol and is thus quite shallow (K_exchange = 1.4 ± 0.2). Thus, the thermodynamics of vectorial copper delivery from the copper chaperone to targets involves small differences in the Cu(I) binding constants of each protein or domain. If the subsequent Cu(I) transfer step to another site in Ccc2, such as the CXC motif (36), is rapid, then each of the copper transfer steps in Fig. 3 would be coupled with the driving force ultimately being provided by ATP hydrolysis.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The copper trafficking pathway between Atx1 and the copper ATPases. Cu(I) (red spheres) is delivered via the metallochaperone Atx1 (green) to the N-terminal domains (purple) of the vesicular copper P-type ATPase Ccc2. In a stepwise mechanism, Cu(I)-Atx1 encounters Ccc2 and forms a transiently docked complex. Copper then rapidly equilibrates between the domains and is pumped into the vesicle driven by the hydrolysis of ATP. In the vesicle, copper ultimately is incorporated into apoproteins such as the multicopper oxidase Fet3.

Because the initial Cu(I) transfer between Atx1 and Ccc2 proceeds rapidly to equilibrium, this step in the cellular trafficking pathway is best considered to be under thermodynamic control. However, the recently established boundary conditions, which indicate an extraordinarily limited cytosolic free copper concentration, need to be considered as the copper chaperone hypothesis is extended. Rae et al. (14) have shown that intracellular free copper concentration is negligible and further suggested that the cytoplasm has a significant overcapacity for copper chelation. This leads to the dilemma of how the copper chaperone can retain Cu(I) in the face of a significant thermodynamic sink if the reaction is under thermodynamic control. Our current data suggest a solution. Copper transfer from chaperone to target is not driven by thermodynamics of the copper-binding sites in these proteins. Rather, Atx1 appears to function as an enzyme; it lowers the kinetic barrier for copper transfer along specific reaction coordinates. In this model, Atx1 catalyzes equilibration of copper between yet to be identified copper donor sites and specific targets such as Ccc2. ATP hydrolysis then drives the compartmentalization of the cytosolic copper available to Atx1. Finally, Atx1 may prevent adventitious copper release by deterring ligand exchange reactions with non-partner proteins, although additional transfer reactions with the latter are required to test this proposal.

Partner Specificity of the Atx1/Ccc2 Reaction-- Given the rapid transfer, we anticipate that a low activation barrier results when the metal-loaded chaperone adopts a preferred orientation with respect to the metal-binding site in the acceptor. In this model, specific intermolecular forces guide the docking interactions and position the metal binding loop in the Cu-donor protein proximal to the unoccupied metal loop in the partner domain. Based on our modeling of both of the ~72-residue N-terminal metal-binding domains of Ccc2 utilizing the coordinates of the NMR structure of Menkes' domain 4 (30), we propose that electrostatic complementation between surfaces on the donor (29, 32) and acceptor proteins will play an important role in achieving the orientation favored for facile metal transfer. The electrostatic properties of the second N-terminal copper-binding domain suggest that it may also serve as a docking site for Atx1. This possibility could increase the likelihood of an encounter between the metallochaperone and its target in vivo and may increase the overall efficiency of copper transport. Once Cu(I) is transferred to either domain of Ccc2, ATP hydrolysis then provides conformational changes that displace the Cu(I) into a vesicle, which is thermodynamically distinct from the cytosol.

As shown by Camakaris and colleagues (37), Cu(I) ions are actively transported through the membrane in an ATP-dependent manner by the Menkes' disease protein, the human homologue of Ccc2. The requirement for active transport is consistent with a vesicular compartment in which the availability of copper (i.e. [Cu(I)]_free) is significantly higher than in the cytosol (Fig. 3). This leads to a further extension of the metallochaperone hypothesis; active transport is required to maintain the gradient of high copper inside the vesicle and low copper in the cytosol. If this is the case, then another copper chaperone may not be required to insert copper into Fet3 within the vesicle; the apo-enzyme could acquire copper by diffusion and collision. A recent report showing that the GEF1 chloride/proton antiporter co-localizes with Fet3 and is required for Fet3 activity (38) raises the interesting possibility that elevated chloride concentrations within these vesicles lead to formation of copper(I) chloride complexes, which can stabilize Cu(I) against disproportionation. This observation is consistent with a compartment that contains elevated levels of free Cu(I) relative to the cytosol.

The facile and reversible nature of the Cu trafficking pathway raises the possibility that the Ccc2-containing secretory vesicles can also serve as a source of copper for cytosolic enzymes. The reverse reaction, copper egress from the vesicle into the cytosol and into Atx1, is allowed by the chemistry, but it remains to be seen whether it is part of the physiology of copper trafficking.

	ACKNOWLEDGEMENTS

We thank R. Pufahl, V. Culotta, J. Widom, A. Rosenzweig, and members of the O'Halloran laboratory for helpful discussions.

	FOOTNOTES

^* This work was supported National Institutes of Health Grant GM54111 (to T. V. O.).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.

^§ Supported by Molecular Toxicology Training Program Postdoctoral Fellowship 5T32ES07284 and by a Gramm Travel Fellowship Award from the Lurie Comprehensive Cancer Center of Northwestern University.

To whom correspondence should be addressed: Dept. of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: t-ohalloran@nwu.edu.

Published, JBC Papers in Press, April 7, 2000, DOI 10.1074/jbc.C000172200

	ABBREVIATIONS

The abbreviations used are: CCS, copper chaperone for superoxide dismutase; DTT, dithiothreitol; GSH, glutathione; MES, 4-morpholineethanesulfonic acid..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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