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Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper Transporter, Ccc2

Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 25, Issue of June 23, pp. 18611–18614, 2000 Accelerated Publication © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. is mediated by the CTR plasma membrane proteins (3– 6). Once Energetics of Copper inside the cell, a portion of the copper is delivered to P-type Trafficking between the ATPases, which pump this metal ion into vesicles for ultimate incorporation into multicopper oxidases. In yeast the P-type Atx1 Metallochaperone ATPase is Ccc2 (7–9) and the multicopper oxidase is Fet3 and the Intracellular (10). Until recently, copper was thought to be delivered to target proteins as glutathione complexes; however, the Atx1 Copper Transporter, Ccc2* metallochaperone protein, a cytosolic Cu(I) receptor, is re- quired downstream of CTR1 and upstream of Ccc2 and related Received for publication, March 16, 2000, and in revised form, ATPases (11, 12). April 5, 2000 Copper chaperone proteins are soluble, intracellular recep- Published, JBC Papers in Press, April 7, 2000, tors that bind and deliver copper to specific partner proteins DOI 10.1074/jbc.C000172200 (12). Initially identified as antioxidant and biosynthetic path- David L. Huffman‡§ and Thomas V. O’Halloran‡¶i way proteins, the yeast metallochaperone Atx1 and the copper From the ‡Department of Chemistry and the chaperone for superoxide dismutase (CCS) have since been ¶Department of Biochemistry, Molecular Biology, and shown to be Cu(I) receptors that interact with specific vesicular Cell Biology, Northwestern University, and cytoplasmic targets, respectively (12–15). Evanston, Illinois 60208-3113 Copper-trafficking pathway proteins including homologues The Atx1 metallochaperone protein is a cytoplasmic of CTR1 (5), Atx1 (16), Cox17 (17), Ccc2 (18 –22), and Fet3 (2, Cu(I) receptor that functions in intracellular copper 23) are highly conserved from yeast to humans. Complementa- trafficking pathways in plants, microbes, and humans. A tion studies demonstrate that human Atx1 (HAH1) functions in key physiological partner of the Saccharomyces cerevi- place of yeast Atx1 (24), and the Wilson’s and Menkes’ disease siae Atx1 is Ccc2, a cation transporting P-type ATPase proteins, homologues of Ccc2, function in place of Ccc2 to de- located in secretory vesicles. Here, we show that Atx1 liver copper to Fet3 (25–27). donates its metal ion cargo to the first N-terminal Atx1- Recent structural studies provide insights into the intracel- like domain of Ccc2 in a direct and reversible manner. lular chemistry of copper transfer (28). Crystallographic stud- The thermodynamic gradient for metal transfer is shal- ies of Atx1 indicate that the metal-binding motif is housed in a low (K 5 1.4 6 0.2), establishing that vectorial exchange surface-exposed loop, with the cysteines located in the first loop delivery of copper by Atx1 is not based on a higher and the first a-helix (29). This fold is conserved in other eu- copper affinity of the target domain. Instead, Atx1 al- karyotic copper-trafficking proteins including Menkes’ ATPase lows rapid metal transfer to its partner. This equilib- domain 4, MNK4 (30), and the CCS metallochaperone domain rium is unaffected by a 50-fold excess of the Cu(I) com- I (31). The two N-terminal domains of Ccc2 are predicted to petitor, glutathione, indicating that Atx1 also protects possess this same babbab-fold, albeit with multiple negatively Cu(I) from nonspecific reactions. Mechanistically, we charged surface residues like MNK4, its human counterpart. In propose that a low activation barrier for transfer be- tween partners results from complementary electro- contrast, Atx1 possesses multiple positively charged lysines on static forces that ultimately orient the metal-binding its surface (29). Mutation of conserved lysines on the surface of loops of Atx1 and Ccc2 for formation of copper-bridged Atx1 greatly reduces the copper-dependent interaction of Atx1 intermediates. These thermodynamic and kinetic con- and Ccc2 in vivo (12, 32). siderations suggest that copper trafficking proteins Cytoplasmic free Cu(I) is notably unavailable to the high overcome the extraordinary copper chelation capacity affinity copper enzymes such as SOD1 (superoxide dismutase) of the eukaryotic cytoplasm by catalyzing the rate of (14). In fact, the results are consistent with the presence of a copper transfer between physiological partners. In this vast excess of specific and nonspecific copper chelation sites sense, metallochaperones work like enzymes, carefully relative to the total number of copper atoms per cell. Further- tailoring energetic barriers along specific reaction path- more, copper exchange rates are generally thought to be slow ways but not others. 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 Copper is an essential cofactor in hydrolytic, electron trans- inappropriate binding sites and yet allow facile release at spe- fer, and oxygen utilization enzymes and is also crucial for high cific destinations. To address these issues, the energetics of affinity iron uptake in yeast (1, 2). Copper uptake in eukaryotes copper transfer between Atx1 and Ccc2 were probed using a direct assay of metal occupancy. * This work was supported National Institutes of Health Grant EXPERIMENTAL PROCEDURES GM54111 (to T. V. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must Cloning and Isolation of Ccc2a and Atx1—The gene encoding the therefore be hereby marked “advertisement” in accordance with 18 72-residue N-terminal Atx1-like domain of Ccc2, Ccc2a, was cloned into U.S.C. Section 1734 solely to indicate this fact. pET11d (Novagen) and resequenced. The resultant plasmid, § Supported by Molecular Toxicology Training Program Postdoctoral pDLHV021, was transformed into Escherichia coli strain BL21(DE3) Fellowship 5T32ES07284 and by a Gramm Travel Fellowship Award and induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside in M9 from the Lurie Comprehensive Cancer Center of Northwestern University. i To whom correspondence should be addressed: Dept. of Chemistry, The abbreviations used are: CCS, copper chaperone for superoxide Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: dismutase; DTT, dithiothreitol; GSH, glutathione; MES, 4-morpho- 847-491-5060; Fax: 847-491-7713; E-mail: [email protected]. lineethanesulfonic acid. This paper is available on line at http://www.jbc.org 18611 This is an Open Access article under the CC BY license. 18612 Energetics of Copper Trafficking between Atx1 and Ccc2 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 Phar- macia 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, concen- trated, 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 hy- 21 21 drolysis (Harvard Microchem) giving e 5 2750 M cm (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 21 21 amino acid hydrolysis (Harvard Microchem, e 5 4950 M cm 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. FIG.1. Copper transfer assay between CuAtx1 and Ccc2a. Pro- Preparation of Protein Samples for Metal Transfer—Frozen protein tein (l,—) and metal ion (E,- - -) concentrations of fractions from strong stocks were treated with excess DTT, washed, and exchanged into the anion exchange chromatography. Controls demonstrate that Ccc2a strongly binds and elutes from the column in fractions 24 –26 (20 m metal insertion buffer immediately prior to loading with copper. Cu(I)- M MES/Na, pH 6, ;0.35 M NaCl) (a), whereas CuAtx1 does not bind to the Atx1 and Cu(I)-Ccc2a were prepared in an N atmosphere chamber column and elutes in fractions 5–7 (20 mM MES/Na, pH 6) (b). c, metal (Vacuum Atmospheres) at 12 °C by adding 1 equivalent of [Cu(I)- ion transfer assay between CuAtx1 and Ccc2a demonstrates that Cu(I) (CH CN) ]PF in CH CN to a solution of apoprotein in 50 mM Tris/MES, 3 4 6 3 is transferred to Ccc2a. 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 Ccc2a binds strongly. ;1, even when the proteins are incubated with excess Cu(I) in the These experiments also provide a quantitative insight into presence of glutathione (GSH) or DTT. The protein samples used for the thermodynamics and kinetics of metal transfer. To test the metal transfer were: Cu(I)-Atx1, 760 mM, Cu/protein ratio 1.0; Cu(I)- equilibrium assumption, the reverse reaction of CuCcc2a with Atx1, 2000 mM, Cu/protein ratio 0.94; Cu(I)-Ccc2a, 1890 mM, Cu/protein apoAtx1 was conducted. As shown in Table I, CuCcc2a can ratio 0.62; apoAtx1, 770 mM; apoCcc2a, 520 mM; apoCcc2a, 2030 mM. transfer metal to apoAtx1. The reverse reaction gives the same Metal concentration was determined by ICP-AES. All samples were stored at 4 °C or 220 °C in an N atmosphere prior to use. product distribution as the forward reaction, establishing that Metal Transfer Assay—Metal ion transfer experiments were per- the system reaches equilibrium in the time frame of the exper- formed by combining Cu(I)-Atx1 or Cu-Ccc2a with apoCcc2a or apoAtx1 iments. Furthermore, the product distribution was independ- in an inert atmosphere maintained at 18 °C at pH 6 in 20 mM MES/Na ent of the incubation time, indicating that copper exchange is or at pH 7.2 in 4 mM NaP . After incubation, the mixture was separated complete even at the shortest possible incubation times (1 min). using the same buffer (pH 6 or pH 7.2 as indicated) with a linear The copper exchange constant (K ) was obtained from gradient (0 – 0.5 M NaCl) on a Bio-Scale Q2 (Biologic) or a RESOURCE exchange Q (1 ml, Amersham Pharmacia Biotech). Column fractions were ana- a linear least squares fit of the data in Table I to the modified lyzed for protein and metal content by the Bradford assay (34) and form of the equilibrium expression shown in Equation 5. Total ICP-AES, respectively. Control experiments indicate routine protein moles of loaded and recovered copper were routinely found in recovery rates of more than 95%. the 90%1 range, and runs with less than 86% recovery were Determination of Equilibrium Constant—The equilibrium expres- not used. The linear least squares fit of the data (correspond- sions for Cu(I) exchange are shown in Equations 1 and 2. ing to experiments 1–14) over a range of protein concentra- @Cu-Atx1] 1 @Ccc2a] º [Atx1] 1 @Cu-Ccc2a] (Eq. 1) eq eq eq eq tions supports the equilibrium assumption implicit in Equa- tion 5 (Fig. 2), revealing a slope (K ) 5 1.4 and standard exchange @Atx1] [Cu-Ccc2a] eq eq K 5 (Eq. 2) deviation s 5 0.1 (correlation coefficient, r, of 0.95). Data for exchange [Cu-Atx1] [Ccc2a] eq eq both the forward and reverse reactions fit equally well, further Using expressions for [Atx1] and [Ccc2a] , indicating that the system is at equilibrium. The averages of eq eq the K values at pH 6.0, 1.5(s 5 0.2), and at pH 7.2, exchange @Atx1] 5 @Atx1] 2 @Cu-Atx1] and (Eq. 3) eq total eq 1.2(s 5 0.3), are the same within experimental error indicating no effect of pH in this range (Fig. 2). @Ccc2a] 5 @Ccc2a] 2 @Cu-Ccc2a] (Eq. 4) eq total eq To determine whether copper competitors can interfere with @Atx1] @Ccc2a] total total the transfer or remove the copper ion from these proteins, we K 5 2 1 2 1 (Eq. 5) exchange F G F G [Cu-Atx1] [Cu-Ccc2a] eq / eq conducted the metal transfer experiment in the presence of a physiologically relevant Cu(I) binding agent, GSH. GSH avidly RESULTS binds Cu(I) to form Cu(GSH) with a stability constant of b 5 2 2 38.8 This in vitro assay of Cu(I) transfer from the metallochaper- 10 (35). The addition of physiological levels of GSH (1–5 mM) one Atx1 to its physiological partner, Ccc2, provides a direct does not effect the final equilibrium position (Table I, experi- test of copper trafficking function. When CuAtx1 is incubated ments 15–17). The copper transfer reaction is unaffected by the with apoCcc2a, the metal partitions between the two proteins presence of even a 50-fold molar excess of GSH over the Cu(I) (Fig. 1). Control experiments demonstrate that Atx1 is not concentration (experiment 17, Table I), indicating that low retained on the Q2 strong anion column (Fig. 1b), whereas molecular weight thiols do not participate in this equilibrium. Energetics of Copper Trafficking between Atx1 and Ccc2 18613 TABLE I Initial Cu(I) a a a b c c Incubation Competitor [Atx1] [Cu-Atx1] [Ccc2a] [Cu-Ccc2a] [Cu(I)-Atx1] [Cu(I)-Ccc2a] K total 0 total 0 eq eq exchange donor min mM mM mM mM mM mM mM 1. Atx1 5 305 305 314 0 120 144 1.31 2. Atx1 5 305 305 314 0 128 159 1.42 3. Atx1 5 305 305 314 0 119 164 1.71 4. Atx1 5 305 305 314 0 126 160 1.48 5. Atx1 5 305 305 209 0 142 119 1.52 6. Atx1 5 305 305 157 0 169 102 1.49 7. Ccc2a 5 90 0 157 97.9 35.3 76.3 1.47 8. Ccc2a 5 90 0 157 97.9 25.7 67.9 1.91 9. Ccc2a 5 90 0 157 97.9 31.4 65.1 1.32 10. Ccc2a 5 90 0 157 97.9 30.5 66.6 1.44 11. Atx1 5 100 94 406 0 18 89 1.28 12. Atx1 5 100 94 406 0 14 83 1.58 13. Atx1 5 200 188 406 0 61 130 1.07 14. Atx1 5 400 376 406 0 198 198 0.97 15. Ccc2a 5 1.1 GSH 90 0 157 97.9 29.0 62.0 1.37 16. Atx1 5 1.5 GSH 100 94 406 0 17 82 1.24 17. Atx1 5 5.0 GSH 100 94 406 0 15 79 1.37 Protein concentrations are normalized to total reaction volume at point of mixing. Copper concentration determined by ICP-AES of protein stock solutions; concentrations are normalized to total volume at point of mixing. Total copper concentration determined from recovery of copper in respective protein-containing fractions after chromatography. d e pH 6.0; pH 7.2. 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. Because the initial Cu(I) transfer between Atx1 and Ccc2 proceeds rapidly to equilibrium, this step in the cellular traf- ficking pathway is best considered to be under thermodynamic control. However, the recently established boundary condi- tions, which indicate an extraordinarily limited cytosolic free FIG.2. Determination of exchange constant, K . The plot copper concentration, need to be considered as the copper chap- exchange of Equation 5 is shown with experiments 1–14 from Table I. A linear erone hypothesis is extended. Rae et al. (14) have shown that regression analysis yields a slope 5 1.4 (corresponding to the equilib- intracellular free copper concentration is negligible and further rium constant, K ) with a standard deviation s 5 0.1 and a exchange m suggested that the cytoplasm has a significant overcapacity for correlation coefficient r 5 0.95. copper chelation. This leads to the dilemma of how the copper chaperone can retain Cu(I) in the face of a significant thermo- We also tested whether Cu-Atx1 could transfer copper to an- dynamic sink if the reaction is under thermodynamic control. other copper-binding protein by incubating 190 mM CuAtx1 Our current data suggest a solution. Copper transfer from anaerobically with 167 mM bovine serum albumin at pH 6 for 5 chaperone to target is not driven by thermodynamics of the min. No copper is transferred from Cu-Atx1 to bovine serum copper-binding sites in these proteins. Rather, Atx1 appears to albumin in four separate determinations. Thus, the strong function as an enzyme; it lowers the kinetic barrier for copper Cu(I) thiolate bonds in Atx1 not only protect Cu(I) from dispro- transfer along specific reaction coordinates. In this model, Atx1 portionation and air oxidation (12) but may also prevent loss of catalyzes equilibration of copper between yet to be identified the metal ion to adventitious binding sites within the cell. copper donor sites and specific targets such as Ccc2. ATP hy- DISCUSSION drolysis then drives the compartmentalization of the cytosolic copper available to Atx1. Finally, Atx1 may prevent adventi- When the copper-bound form of the chaperone is directly tious copper release by deterring ligand exchange reactions incubated with apoCcc2a, Cu(I) equilibrates between the two with non-partner proteins, although additional transfer reac- proteins, and it does so rapidly. A stepwise mechanism for tions with the latter are required to test this proposal. direct and rapid metal transfer is proposed. In the first step, Partner Specificity of the Atx1/Ccc2 Reaction—Given the CuAtx1 docks apoCcc2a. A specific orientation between CuAtx1 and apoCcc2a could poise the Cu(I) center for nucleophilic rapid transfer, we anticipate that a low activation barrier re- sults when the metal-loaded chaperone adopts a preferred ori- attack by thiol from the adjacent protein, forming a Cu(S-Cys) intermediate. In the next step, the copper rapidly partitions entation with respect to the metal-binding site in the acceptor. In this model, specific intermolecular forces guide the docking between the two metal-binding sites within the protein-protein complex via the formation and decay of two- and three-coordi- interactions and position the metal binding loop in the Cu- donor protein proximal to the unoccupied metal loop in the nate copper thiolate intermediates. In the final step, the com- plex dissociates to provide apoAtx1 and Cu-Ccc2a. partner domain. Based on our modeling of both of the ;72- Thermodynamic versus Kinetic Control of Copper Traffick- residue N-terminal metal-binding domains of Ccc2 utilizing the ing—The metal exchange results shown herein demonstrate coordinates of the NMR structure of Menkes’ domain 4 (30), we that the thermodynamic gradient for copper transfer between propose that electrostatic complementation between surfaces this copper chaperone and its target domains is ;0.2 kcal/mol on the donor (29, 32) and acceptor proteins will play an impor- and is thus quite shallow (K 5 1.4 6 0.2). Thus, the tant role in achieving the orientation favored for facile metal exchange 18614 Energetics of Copper Trafficking between Atx1 and Ccc2 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. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632–2636 transfer. The electrostatic properties of the second N-terminal 9. Yuan, D. S., Dancis, A., and Klausner, R. D. (1997) J. Biol. Chem. 272, copper-binding domain suggest that it may also serve as a 25787–25793 docking site for Atx1. This possibility could increase the like- 10. Askwith, C., Eide, D., Van Ho, A., P. S., B., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403– 410 lihood of an encounter between the metallochaperone and its 11. Lin, S.-J., Pufahl, R. A., Dancis, A., O’Halloran, T. V., and Culotta, V. C. (1997) target in vivo and may increase the overall efficiency of copper J. Biol. 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Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper Transporter, Ccc2

Huffman, David L.; O'Halloran, Thomas V.
Journal of Biological Chemistry – Jun 1, 2000
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 25, Issue of June 23, pp. 18611–18614, 2000 Accelerated Publication © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. is mediated by the CTR plasma membrane proteins (3– 6). Once Energetics of Copper inside the cell, a portion of the copper is delivered to P-type Trafficking between the ATPases, which pump this metal ion into vesicles for ultimate incorporation into multicopper oxidases. In yeast the P-type Atx1 Metallochaperone ATPase is Ccc2 (7–9) and the multicopper oxidase is Fet3 and the Intracellular (10). Until recently, copper was thought to be delivered to target proteins as glutathione complexes; however, the Atx1 Copper Transporter, Ccc2* metallochaperone protein, a cytosolic Cu(I) receptor, is re- quired downstream of CTR1 and upstream of Ccc2 and related Received for publication, March 16, 2000, and in revised form, ATPases (11, 12). April 5, 2000 Copper chaperone proteins are soluble, intracellular recep- Published, JBC Papers in Press, April 7, 2000, tors that bind and deliver copper to specific partner proteins DOI 10.1074/jbc.C000172200 (12). Initially identified as antioxidant and biosynthetic path- David L. Huffman‡§ and Thomas V. O’Halloran‡¶i way proteins, the yeast metallochaperone Atx1 and the copper From the ‡Department of Chemistry and the chaperone for superoxide dismutase (CCS) have since been ¶Department of Biochemistry, Molecular Biology, and shown to be Cu(I) receptors that interact with specific vesicular Cell Biology, Northwestern University, and cytoplasmic targets, respectively (12–15). Evanston, Illinois 60208-3113 Copper-trafficking pathway proteins including homologues The Atx1 metallochaperone protein is a cytoplasmic of CTR1 (5), Atx1 (16), Cox17 (17), Ccc2 (18 –22), and Fet3 (2, Cu(I) receptor that functions in intracellular copper 23) are highly conserved from yeast to humans. Complementa- trafficking pathways in plants, microbes, and humans. A tion studies demonstrate that human Atx1 (HAH1) functions in key physiological partner of the Saccharomyces cerevi- place of yeast Atx1 (24), and the Wilson’s and Menkes’ disease siae Atx1 is Ccc2, a cation transporting P-type ATPase proteins, homologues of Ccc2, function in place of Ccc2 to de- located in secretory vesicles. Here, we show that Atx1 liver copper to Fet3 (25–27). donates its metal ion cargo to the first N-terminal Atx1- Recent structural studies provide insights into the intracel- like domain of Ccc2 in a direct and reversible manner. lular chemistry of copper transfer (28). Crystallographic stud- The thermodynamic gradient for metal transfer is shal- ies of Atx1 indicate that the metal-binding motif is housed in a low (K 5 1.4 6 0.2), establishing that vectorial exchange surface-exposed loop, with the cysteines located in the first loop delivery of copper by Atx1 is not based on a higher and the first a-helix (29). This fold is conserved in other eu- copper affinity of the target domain. Instead, Atx1 al- karyotic copper-trafficking proteins including Menkes’ ATPase lows rapid metal transfer to its partner. This equilib- domain 4, MNK4 (30), and the CCS metallochaperone domain rium is unaffected by a 50-fold excess of the Cu(I) com- I (31). The two N-terminal domains of Ccc2 are predicted to petitor, glutathione, indicating that Atx1 also protects possess this same babbab-fold, albeit with multiple negatively Cu(I) from nonspecific reactions. Mechanistically, we charged surface residues like MNK4, its human counterpart. In propose that a low activation barrier for transfer be- tween partners results from complementary electro- contrast, Atx1 possesses multiple positively charged lysines on static forces that ultimately orient the metal-binding its surface (29). Mutation of conserved lysines on the surface of loops of Atx1 and Ccc2 for formation of copper-bridged Atx1 greatly reduces the copper-dependent interaction of Atx1 intermediates. These thermodynamic and kinetic con- and Ccc2 in vivo (12, 32). siderations suggest that copper trafficking proteins Cytoplasmic free Cu(I) is notably unavailable to the high overcome the extraordinary copper chelation capacity affinity copper enzymes such as SOD1 (superoxide dismutase) of the eukaryotic cytoplasm by catalyzing the rate of (14). In fact, the results are consistent with the presence of a copper transfer between physiological partners. In this vast excess of specific and nonspecific copper chelation sites sense, metallochaperones work like enzymes, carefully relative to the total number of copper atoms per cell. Further- tailoring energetic barriers along specific reaction path- more, copper exchange rates are generally thought to be slow ways but not others. 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 Copper is an essential cofactor in hydrolytic, electron trans- inappropriate binding sites and yet allow facile release at spe- fer, and oxygen utilization enzymes and is also crucial for high cific destinations. To address these issues, the energetics of affinity iron uptake in yeast (1, 2). Copper uptake in eukaryotes copper transfer between Atx1 and Ccc2 were probed using a direct assay of metal occupancy. * This work was supported National Institutes of Health Grant EXPERIMENTAL PROCEDURES GM54111 (to T. V. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must Cloning and Isolation of Ccc2a and Atx1—The gene encoding the therefore be hereby marked “advertisement” in accordance with 18 72-residue N-terminal Atx1-like domain of Ccc2, Ccc2a, was cloned into U.S.C. Section 1734 solely to indicate this fact. pET11d (Novagen) and resequenced. The resultant plasmid, § Supported by Molecular Toxicology Training Program Postdoctoral pDLHV021, was transformed into Escherichia coli strain BL21(DE3) Fellowship 5T32ES07284 and by a Gramm Travel Fellowship Award and induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside in M9 from the Lurie Comprehensive Cancer Center of Northwestern University. i To whom correspondence should be addressed: Dept. of Chemistry, The abbreviations used are: CCS, copper chaperone for superoxide Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Tel.: dismutase; DTT, dithiothreitol; GSH, glutathione; MES, 4-morpho- 847-491-5060; Fax: 847-491-7713; E-mail: [email protected]. lineethanesulfonic acid. This paper is available on line at http://www.jbc.org 18611 This is an Open Access article under the CC BY license. 18612 Energetics of Copper Trafficking between Atx1 and Ccc2 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 Phar- macia 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, concen- trated, 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 hy- 21 21 drolysis (Harvard Microchem) giving e 5 2750 M cm (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 21 21 amino acid hydrolysis (Harvard Microchem, e 5 4950 M cm 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. FIG.1. Copper transfer assay between CuAtx1 and Ccc2a. Pro- Preparation of Protein Samples for Metal Transfer—Frozen protein tein (l,—) and metal ion (E,- - -) concentrations of fractions from strong stocks were treated with excess DTT, washed, and exchanged into the anion exchange chromatography. Controls demonstrate that Ccc2a strongly binds and elutes from the column in fractions 24 –26 (20 m metal insertion buffer immediately prior to loading with copper. Cu(I)- M MES/Na, pH 6, ;0.35 M NaCl) (a), whereas CuAtx1 does not bind to the Atx1 and Cu(I)-Ccc2a were prepared in an N atmosphere chamber column and elutes in fractions 5–7 (20 mM MES/Na, pH 6) (b). c, metal (Vacuum Atmospheres) at 12 °C by adding 1 equivalent of [Cu(I)- ion transfer assay between CuAtx1 and Ccc2a demonstrates that Cu(I) (CH CN) ]PF in CH CN to a solution of apoprotein in 50 mM Tris/MES, 3 4 6 3 is transferred to Ccc2a. 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 Ccc2a binds strongly. ;1, even when the proteins are incubated with excess Cu(I) in the These experiments also provide a quantitative insight into presence of glutathione (GSH) or DTT. The protein samples used for the thermodynamics and kinetics of metal transfer. To test the metal transfer were: Cu(I)-Atx1, 760 mM, Cu/protein ratio 1.0; Cu(I)- equilibrium assumption, the reverse reaction of CuCcc2a with Atx1, 2000 mM, Cu/protein ratio 0.94; Cu(I)-Ccc2a, 1890 mM, Cu/protein apoAtx1 was conducted. As shown in Table I, CuCcc2a can ratio 0.62; apoAtx1, 770 mM; apoCcc2a, 520 mM; apoCcc2a, 2030 mM. transfer metal to apoAtx1. The reverse reaction gives the same Metal concentration was determined by ICP-AES. All samples were stored at 4 °C or 220 °C in an N atmosphere prior to use. product distribution as the forward reaction, establishing that Metal Transfer Assay—Metal ion transfer experiments were per- the system reaches equilibrium in the time frame of the exper- formed by combining Cu(I)-Atx1 or Cu-Ccc2a with apoCcc2a or apoAtx1 iments. Furthermore, the product distribution was independ- in an inert atmosphere maintained at 18 °C at pH 6 in 20 mM MES/Na ent of the incubation time, indicating that copper exchange is or at pH 7.2 in 4 mM NaP . After incubation, the mixture was separated complete even at the shortest possible incubation times (1 min). using the same buffer (pH 6 or pH 7.2 as indicated) with a linear The copper exchange constant (K ) was obtained from gradient (0 – 0.5 M NaCl) on a Bio-Scale Q2 (Biologic) or a RESOURCE exchange Q (1 ml, Amersham Pharmacia Biotech). Column fractions were ana- a linear least squares fit of the data in Table I to the modified lyzed for protein and metal content by the Bradford assay (34) and form of the equilibrium expression shown in Equation 5. Total ICP-AES, respectively. Control experiments indicate routine protein moles of loaded and recovered copper were routinely found in recovery rates of more than 95%. the 90%1 range, and runs with less than 86% recovery were Determination of Equilibrium Constant—The equilibrium expres- not used. The linear least squares fit of the data (correspond- sions for Cu(I) exchange are shown in Equations 1 and 2. ing to experiments 1–14) over a range of protein concentra- @Cu-Atx1] 1 @Ccc2a] º [Atx1] 1 @Cu-Ccc2a] (Eq. 1) eq eq eq eq tions supports the equilibrium assumption implicit in Equa- tion 5 (Fig. 2), revealing a slope (K ) 5 1.4 and standard exchange @Atx1] [Cu-Ccc2a] eq eq K 5 (Eq. 2) deviation s 5 0.1 (correlation coefficient, r, of 0.95). Data for exchange [Cu-Atx1] [Ccc2a] eq eq both the forward and reverse reactions fit equally well, further Using expressions for [Atx1] and [Ccc2a] , indicating that the system is at equilibrium. The averages of eq eq the K values at pH 6.0, 1.5(s 5 0.2), and at pH 7.2, exchange @Atx1] 5 @Atx1] 2 @Cu-Atx1] and (Eq. 3) eq total eq 1.2(s 5 0.3), are the same within experimental error indicating no effect of pH in this range (Fig. 2). @Ccc2a] 5 @Ccc2a] 2 @Cu-Ccc2a] (Eq. 4) eq total eq To determine whether copper competitors can interfere with @Atx1] @Ccc2a] total total the transfer or remove the copper ion from these proteins, we K 5 2 1 2 1 (Eq. 5) exchange F G F G [Cu-Atx1] [Cu-Ccc2a] eq / eq conducted the metal transfer experiment in the presence of a physiologically relevant Cu(I) binding agent, GSH. GSH avidly RESULTS binds Cu(I) to form Cu(GSH) with a stability constant of b 5 2 2 38.8 This in vitro assay of Cu(I) transfer from the metallochaper- 10 (35). The addition of physiological levels of GSH (1–5 mM) one Atx1 to its physiological partner, Ccc2, provides a direct does not effect the final equilibrium position (Table I, experi- test of copper trafficking function. When CuAtx1 is incubated ments 15–17). The copper transfer reaction is unaffected by the with apoCcc2a, the metal partitions between the two proteins presence of even a 50-fold molar excess of GSH over the Cu(I) (Fig. 1). Control experiments demonstrate that Atx1 is not concentration (experiment 17, Table I), indicating that low retained on the Q2 strong anion column (Fig. 1b), whereas molecular weight thiols do not participate in this equilibrium. Energetics of Copper Trafficking between Atx1 and Ccc2 18613 TABLE I Initial Cu(I) a a a b c c Incubation Competitor [Atx1] [Cu-Atx1] [Ccc2a] [Cu-Ccc2a] [Cu(I)-Atx1] [Cu(I)-Ccc2a] K total 0 total 0 eq eq exchange donor min mM mM mM mM mM mM mM 1. Atx1 5 305 305 314 0 120 144 1.31 2. Atx1 5 305 305 314 0 128 159 1.42 3. Atx1 5 305 305 314 0 119 164 1.71 4. Atx1 5 305 305 314 0 126 160 1.48 5. Atx1 5 305 305 209 0 142 119 1.52 6. Atx1 5 305 305 157 0 169 102 1.49 7. Ccc2a 5 90 0 157 97.9 35.3 76.3 1.47 8. Ccc2a 5 90 0 157 97.9 25.7 67.9 1.91 9. Ccc2a 5 90 0 157 97.9 31.4 65.1 1.32 10. Ccc2a 5 90 0 157 97.9 30.5 66.6 1.44 11. Atx1 5 100 94 406 0 18 89 1.28 12. Atx1 5 100 94 406 0 14 83 1.58 13. Atx1 5 200 188 406 0 61 130 1.07 14. Atx1 5 400 376 406 0 198 198 0.97 15. Ccc2a 5 1.1 GSH 90 0 157 97.9 29.0 62.0 1.37 16. Atx1 5 1.5 GSH 100 94 406 0 17 82 1.24 17. Atx1 5 5.0 GSH 100 94 406 0 15 79 1.37 Protein concentrations are normalized to total reaction volume at point of mixing. Copper concentration determined by ICP-AES of protein stock solutions; concentrations are normalized to total volume at point of mixing. Total copper concentration determined from recovery of copper in respective protein-containing fractions after chromatography. d e pH 6.0; pH 7.2. 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. Because the initial Cu(I) transfer between Atx1 and Ccc2 proceeds rapidly to equilibrium, this step in the cellular traf- ficking pathway is best considered to be under thermodynamic control. However, the recently established boundary condi- tions, which indicate an extraordinarily limited cytosolic free FIG.2. Determination of exchange constant, K . The plot copper concentration, need to be considered as the copper chap- exchange of Equation 5 is shown with experiments 1–14 from Table I. A linear erone hypothesis is extended. Rae et al. (14) have shown that regression analysis yields a slope 5 1.4 (corresponding to the equilib- intracellular free copper concentration is negligible and further rium constant, K ) with a standard deviation s 5 0.1 and a exchange m suggested that the cytoplasm has a significant overcapacity for correlation coefficient r 5 0.95. copper chelation. This leads to the dilemma of how the copper chaperone can retain Cu(I) in the face of a significant thermo- We also tested whether Cu-Atx1 could transfer copper to an- dynamic sink if the reaction is under thermodynamic control. other copper-binding protein by incubating 190 mM CuAtx1 Our current data suggest a solution. Copper transfer from anaerobically with 167 mM bovine serum albumin at pH 6 for 5 chaperone to target is not driven by thermodynamics of the min. No copper is transferred from Cu-Atx1 to bovine serum copper-binding sites in these proteins. Rather, Atx1 appears to albumin in four separate determinations. Thus, the strong function as an enzyme; it lowers the kinetic barrier for copper Cu(I) thiolate bonds in Atx1 not only protect Cu(I) from dispro- transfer along specific reaction coordinates. In this model, Atx1 portionation and air oxidation (12) but may also prevent loss of catalyzes equilibration of copper between yet to be identified the metal ion to adventitious binding sites within the cell. copper donor sites and specific targets such as Ccc2. ATP hy- DISCUSSION drolysis then drives the compartmentalization of the cytosolic copper available to Atx1. Finally, Atx1 may prevent adventi- When the copper-bound form of the chaperone is directly tious copper release by deterring ligand exchange reactions incubated with apoCcc2a, Cu(I) equilibrates between the two with non-partner proteins, although additional transfer reac- proteins, and it does so rapidly. A stepwise mechanism for tions with the latter are required to test this proposal. direct and rapid metal transfer is proposed. In the first step, Partner Specificity of the Atx1/Ccc2 Reaction—Given the CuAtx1 docks apoCcc2a. A specific orientation between CuAtx1 and apoCcc2a could poise the Cu(I) center for nucleophilic rapid transfer, we anticipate that a low activation barrier re- sults when the metal-loaded chaperone adopts a preferred ori- attack by thiol from the adjacent protein, forming a Cu(S-Cys) intermediate. In the next step, the copper rapidly partitions entation with respect to the metal-binding site in the acceptor. In this model, specific intermolecular forces guide the docking between the two metal-binding sites within the protein-protein complex via the formation and decay of two- and three-coordi- interactions and position the metal binding loop in the Cu- donor protein proximal to the unoccupied metal loop in the nate copper thiolate intermediates. In the final step, the com- plex dissociates to provide apoAtx1 and Cu-Ccc2a. partner domain. Based on our modeling of both of the ;72- Thermodynamic versus Kinetic Control of Copper Traffick- residue N-terminal metal-binding domains of Ccc2 utilizing the ing—The metal exchange results shown herein demonstrate coordinates of the NMR structure of Menkes’ domain 4 (30), we that the thermodynamic gradient for copper transfer between propose that electrostatic complementation between surfaces this copper chaperone and its target domains is ;0.2 kcal/mol on the donor (29, 32) and acceptor proteins will play an impor- and is thus quite shallow (K 5 1.4 6 0.2). Thus, the tant role in achieving the orientation favored for facile metal exchange 18614 Energetics of Copper Trafficking between Atx1 and Ccc2 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. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632–2636 transfer. The electrostatic properties of the second N-terminal 9. Yuan, D. S., Dancis, A., and Klausner, R. D. (1997) J. Biol. Chem. 272, copper-binding domain suggest that it may also serve as a 25787–25793 docking site for Atx1. This possibility could increase the like- 10. Askwith, C., Eide, D., Van Ho, A., P. S., B., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403– 410 lihood of an encounter between the metallochaperone and its 11. Lin, S.-J., Pufahl, R. A., Dancis, A., O’Halloran, T. V., and Culotta, V. C. (1997) target in vivo and may increase the overall efficiency of copper J. Biol. 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