Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone*

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (Ka) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are ∼105 to 106 m–1, suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and Ka values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1–6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.

localized in the trans-Golgi network of hepatocytes where it delivers copper to key metalloenzymes, including ceruloplasmin, a multicopper oxidase involved in high affinity iron uptake (4,5). This process is dependent on interaction with Atox1, a copper chaperone that delivers Cu(I) ions to WND (6,7). At elevated copper concentrations, WND redistributes to cytoplasmic vesicles and mediates copper efflux from the cell (4,8). Similarly, the Menkes disease protein (MNK) relocalizes from the trans-Golgi network to the plasma membrane in response to potentially toxic copper concentrations (9,10).
Both proteins consist of eight transmembrane domains, of which the sixth contains an invariant CPC motif proposed to bind metal ions. Phosphatase-and ATP-binding domains and a phosphorylation site are conserved as well (11). Metal transport ATPases are distinguished from other P-type ATPases by the presence of multiple soluble N-terminal domains containing a conserved CXXC metal-binding motif (12) (Fig. 1). Both WND and MNK contain six such domains, whereas the yeast homolog, Ccc2 (13), contains only two repeats and the Drosophila melanogaster homolog contains four. These domains are present not only in the copper ATPases but also occur in Zn(II), Cd(II), and Pb(II) transporters (11,12). The Atox1 metallochaperone and its homologs, both eukaryotic and prokaryotic, contain a single CXXC motif (14). Crystal and solution structures of Atox1 (15), yeast Atx1 (16,17), bacterial CopZ (18,19), and single domains of MNK (20) and Ccc2 (21) reveal a conserved ␤␣␤␤␣␤-fold with the cysteines from the CXXC motif coordinating metal ions on a surface-exposed loop. Notably, the CXXC motifs from two Atox1 molecules coordinate a single Cu(I) ion in the x-ray structure (15). Although there are no structures of polypeptides comprising all six repeats from WND or MNK, their metal-binding properties have been studied in some detail. Both the WND and MNK N termini bind 5-6 copper ions (22), and x-ray absorption spectroscopic studies indicate that the copper is present as Cu(I), ligated by the sulfurs from two cysteines (23)(24)(25).
The roles of the six WND and MNK metal-binding domains in copper metabolism are not well established. Numerous genetic and biochemical studies suggest that the domains are not functionally equivalent. For WND, yeast complementation assays indicate that the sixth domain alone is sufficient for copper loading of Fet3, the yeast homolog of ceruloplasmin (26,27). Furthermore, the second or third metal-binding domain cannot substitute for the sixth domain, supporting distinct functions for the individual domains (27). Recently, a more specific function has been assigned to domains 5 and 6. These domains are responsible for the cooperative effect of copper on WND catalytic phosphorylation activity (28). In contrast to WND, yeast complementation studies on MNK indicate that the first four N-terminal domains are important for copper transport (29). It may be that the domains function differently in the two ATPases. Another potential function for the metalbinding domains is mediating copper-responsive cellular relocalization. For example, mutations in MNK domains 4 -6 interfere with the ability to traffic to the plasma membrane in response to elevated copper (30,31). Finally, some of the domains may interact specifically with the copper chaperone Atox1. According to yeast two-hybrid assays using both WND (32) and MNK (33), Atox1 interacts most strongly with domains 1-4 and not at all with domains 5-6, although an interaction with MNK domains 5-6 has been detected by surface plasmon resonance (33). These interactions are metal-dependent and specific for copper.
Taken together, these data suggest that the six domains play discrete roles in the function and regulation of WND. The metal-binding domains most important for interaction with Atox1 (domains 1-4) are clearly distinct from those required for copper transport (domains 5-6). Domains 1-4 of WND have also been proposed to function in copper-responsive localization (27,28). Although all six metal-binding domains can bind copper (22,34), their copper-binding affinities are not known. Furthermore, the copper-binding affinity of Atox1 has not been reported. Such quantitative data are crucial to understanding the functions of individual domains. We have used isothermal titration calorimetry (ITC) to measure the association constant (K a ) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to Atox1. These data provide new insight into the energetics of copper transfer and into how the individual functions of the WND domains are defined.

EXPERIMENTAL PROCEDURES
Protein Cloning and Expression-Plasmids containing the cDNA for the entire WND and for Atox1 were generously supplied by Dr. Jonathan Gitlin (Washington University School of Medicine). Individual constructs comprising domains 1-2 (WD12), domains 3-4 (WD34), domains 5-6 (WD56), domains 1-4 (WD14), and domains 1-6 (WD16) were generated by PCR using the primers listed in Table I. The PCR products for the WND proteins were gel-purified and inserted into the pET32 Xa/LIC vector following the ligation independent cloning protocol from Novagen. This vector includes an N-terminal thioredoxin tag followed by a His 6 tag, a thrombin cleavage site, an S-tag, and a Factor Xa cleavage site. Plasmids were then transformed into BL21(DE3)pLysS cells for induction tests. The primers for Atox1 incorporate an NdeI sequence at the N terminus and a stop codon and EcoRI sequence at the C terminus. The Atox1 PCR product was gel-purified and cloned into the pET21b vector (Novagen) using conventional methods.
For the expression of WD12, WD34, WD56, and WD14, a single colony was used to inoculate a 50-or 100-ml culture of LB medium containing 1 mg/ml carbenicillin and 0.4 mg/ml chloramphenicol (LB/ carb/chlor). After incubating overnight, 8 ml of this culture was used to inoculate multiple 2-liter flasks containing 1 liter of LB/carb/chlor, and these flasks were incubated at 250 rpm and 37°C. When the optical density at 600 nm (A 600 ) reached 0.6 -0.8, protein expression was induced by the addition of 0.9 -1.0 mM isopropyl-␤-D-thiogalactopyranoside. Three hours later cells were harvested by centrifugation at 5000 ϫ g for 20 min and stored at Ϫ80°C. The procedure for WD16 was similar, but the temperature was reduced to 16°C at an A 600 of ϳ0.3. Protein expression was induced with 0.9 mM isopropyl-␤-D-thiogalactopyranoside at an A 600 of 0.7-0.9, and the cells were harvested after 20 h. This The six N-terminal CXXC-containing metal-binding domains (MBD1-6) acquire copper from the Atox1 chaperone. The protein is predicted to have eight transmembrane domains that form a channel for copper translocation. Two cysteines in the transmembrane domain 6 CPC motif are believed to bind copper. Highly conserved motifs are also found in the ATP-binding and phosphatase domains.
protocol was necessary for optimal solubility of WD16. Atox1 was expressed using the same procedures as for WD12, WD34, WD56, and WD14 except that chloramphenicol was not included in the medium.
Protein Purification-For purification of WD12, WD34, WD56, and WD14, cells were thawed in 50 mM Hepes, pH 7.5, 500 mM NaCl, and 5% glycerol (buffer A). EDTA-free protease inhibitor tablets (Roche Applied Science) and a small amount of solid DNase I were added, and protein was extracted with stirring for 30 min to 1 h at room temperature. This solution was then centrifuged at 9000 ϫ g for 30 min, and the supernatant was applied to a 20-ml pre-equilibrated, nickel-loaded chelating-Sepharose column (Amersham Biosciences), rinsed with buffer A, and eluted with an 8-column volume gradient of 300 mM imidazole, pH 7.5, and 500 mM NaCl (buffer B). For WD12, WD34, and WD56, fractions containing the tagged protein of interest were pooled and dialyzed versus 2 liters of buffer A overnight at 4°C to remove excess imidazole. To cleave all three affinity tags, 200 units of Factor Xa (Novagen) were added, and the mixture was incubated at 18°C for 16 h. At this point, 1 mM phenylmethylsulfonyl fluoride was added to stop the proteolytic cleavage. For WD14, the tagged protein from the nickel column was concentrated, cleaved with 100 units of Factor Xa, and then diluted to remove excess imidazole.
Cleavage reactions were reloaded onto the nickel column, rinsed with buffer A, and eluted with a 6-column volume gradient into buffer B. Cleaved protein without the tags eluted at ϳ24 mM imidazole except WD14 and WD16, which required up to 90 mM imidazole, whereas the uncleaved, tagged protein and the cleaved tags eluted at ϳ150 mM imidazole. Fractions containing the cleaved protein were concentrated with a Centriprep 10 (Amicon) to 5 ml, loaded onto a Superdex 75 gel filtration column (Amersham Biosciences), and eluted with buffer A. For WD14, 1 mM EDTA and 5 mM dithiothreitol were added to buffer A for this step. Fractions Ͼ95% pure by SDS-PAGE were pooled, concentrated to 10 mg/ml, flash-frozen in liquid nitrogen in small aliquots, and stored at Ϫ80°C. The purification of WD16 was performed according to the same procedures, with the following modifications to prevent proteolysis and protein precipitation. First, 500 l of EDTA-free protease inhibitor mixture in Me 2 SO (Sigma) was added during the extraction step. Second, fractions from the nickel column were eluted into test tubes containing buffer A supplemented with 1 mM phenylmethylsulfonyl fluoride, diluting the fractions 1:1. Third, excess imidazole was removed using a Superdex 200 gel filtration column. Finally, the Factor Xa cleavage was carried out for just 12 h. For all proteins, successful removal of the three affinity tags was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis using a Bruker Reflex III instrument in the Mass Spectrometry Facility, Department of Chemistry, University of Arizona. Purification of Atox1 was initiated by three rounds of freezing and thawing the cells followed by extraction into 500 ml of 20 mM MES, pH 6.0, for 1 h. The cell extract was centrifuged at 9000 ϫ g for 50 min, and the supernatant was loaded onto an S column (Amersham Biosciences) and eluted with a 3-column volume gradient into 20 mM MES, pH 6.0, and 500 mM NaCl. The protein was then concentrated to 20 ml with a Centricon Plus-20 5000 molecular weight cut off (Amicon), applied in 3-ml aliquots to a Superdex 75 gel filtration column, and eluted with 20 mM MES, pH 6.0, and 200 mM NaCl. Fractions containing pure protein were pooled, frozen in liquid nitrogen, and stored at Ϫ80°C.
Isothermal Titration Calorimetry Measurements-Starting stock solution protein concentrations were measured using extinction coeffi-cients at 280 nm, determined by amino acid hydrolysis (WD12, 25,588 19,840 M Ϫ1 cm Ϫ1 ; and Atox1, 3884 M Ϫ1 cm Ϫ1 ). Protein was concentrated or diluted to 50 M in 3-ml amounts, and 10 mM EDTA was added. This stock protein solution was incubated for 30 min on ice and then transferred to a Slide-A-Lyzer dialysis cassette (Pierce) with a 3000-Da molecular mass cutoff. After three successive rounds of dialysis versus 500 ml of chelexed 100 mM MES, pH 6.5, 100 mM NaCl, and 0.6 mM tris-2-carboxyethylphosphine, the concentration of EDTA was estimated to be ϳ0.36 M. The sample was then transferred to a Coy anaerobic chamber and dialyzed three times versus 500 ml degassed, chelexed 100 mM MES, pH 6.5, and 100 mM NaCl (buffer M), at which point the concentration of tris-2-carboxyethylphosphine was estimated to be ϳ130 pM. After this treatment, the residual copper content was determined by atomic absorption spectroscopy using a PerkinElmer Life Sciences AAnalyst 700 with a high performance flame (detection limit for copper, 1.5 ppb) and copper lumina hollow cathode lamp. A standard curve was generated using a copper atomic absorption standard (Aldrich). All the samples contained ϳ30 ppb copper as did buffer M. Taking into account the protein concentration, the average copper content prior to the ITC experiments was 0.03, 0.04, 0.06, 0.05, 0.03, and 0.09 copper ions per protein molecule for Atox1, WD12, WD34, WD56, WD14, and WD16, respectively. The number of reduced cysteines was measured using the thiol and sulfide quantitation kit from Molecular Probes.
A stock solution of 9 mM CuCl in 10 mM HCl and 1.0 M NaCl was degassed extensively on a vacuum line, backfilled with argon, and stored in the anaerobic chamber. The concentration of this stock solution was determined by inductively coupled plasma optical emission spectroscopic analysis by Galbraith Laboratories (Knoxville, TN). For all copper-binding experiments both protein and copper stock solutions were diluted with buffer M immediately before the run. Appropriate protein and copper concentrations for each titration were determined empirically and ranged from 3 to 25 M protein and 250 to 600 M Cu(I). Samples were checked by dynamic light scattering to ensure that the addition of Cu(I) did not cause aggregation. A sample of the diluted protein was set aside to determine the final concentration for each experiment. Because the protein concentration was too low to measure the absorbance at 280 nm, the enhanced bicinchoninic acid protein assay (Pierce) was used. The protein was removed from the anaerobic chamber in a 2.5-ml Hamilton gas-tight syringe and loaded into the calorimeter (MicroCal) sample cell under a constant pool of high grade pure argon. The Cu(I) solution was then loaded into a 250-l titration syringe in the anaerobic chamber and transferred to the calorimeter chamber under argon gas.
At this point, the experiment was initiated. In a typical run, an automated sequence of 30 injections of 6 -10 l of Cu(I) were applied to the protein in the sample cell with 6 min between injections to allow equilibration. The reaction solution was stirred at 300 rpm, and the temperature of the chamber was maintained at 22°C, working against a surrounding water bath temperature of 15°C. All experiments were repeated at least three times. All data were analyzed with the Origin 5.0 software package from MicroCal, using either a one-or two-sitebinding model. A background correction was applied to each experiment, corresponding to the average of the last four injections. This value was consistent with control experiments in which the Cu(I) solution was titrated into buffer alone. After subtraction of this heat of dilution, a nonlinear least squares method was used to minimize 2 values and obtain best fit parameters for the number of binding sites, n, the association constant, K a , and the change in enthalpy, ⌬H°. The specificity of copper binding was addressed by two additional negative

RESULTS
ITC is an excellent technique for determining the thermodynamic properties of a reaction under defined solution conditions. Measuring the heat generated or absorbed on ligand binding to a protein can yield values for the stoichiometry, n, as well as for K a , ⌬H°, ⌬G°, and ⌬S°. To obtain useful ITC data, every aspect of the experiment must be considered carefully (35,36). Studies of WND and Atox1 are further complicated by the presence of multiple cysteine residues, oxidation of which can cause aggregation, and by the air sensitivity and low solubility of Cu(I). As described above, we have developed protocols for preparing both the protein sample and the Cu(I) titrant solution for loading the calorimeter cell and for conducting the titration anaerobically. The validity of these procedures was tested by titrating Mg(II) into Atox1 (Fig. 2) and WD12, which do not bind Mg(II), and by titrating Cu(I) into lysozyme (Fig. 3), which does not bind Cu(I) specifically. None of these control experiments showed specific Cu(I) binding. By contrast, we obtained interpretable and reproducible data for Cu(I) binding to the WND metal-binding domains and Atox1. In each experiment, titrations of Cu(I) into solutions of metal-free, reduced protein resulted in large exothermic peaks, which eventually diminished to just the heat of dilution (see Figs. 4 -9).
Data for Atox1 (Fig. 4), WD34 (Fig. 5), and WD56 (Fig. 6) were fitted well with a one-site binding model. The n value of 1.41 Ϯ 0.22 for Atox1 is consistent with binding a single Cu(I) ion. For both WD34 and WD56, n values very close to 2 were obtained, indicating that each CXXC motif in these polypeptides can bind a Cu(I) ion. The K a values for WD34 and WD56, ϳ10 6 M Ϫ1 , are higher than that for Atox1, ϳ10 5 M Ϫ1 . In particular, WD34 binds Cu(I) approximately an order of magnitude more tightly than the chaperone (Table II). Consideration of the experimental error for each measurement suggests that the binding constants are roughly similar, however. Fitting the data for WD12 (Fig. 7) with a one-site model yielded a stoichiometry of 0.95 Ϯ 0.38 Cu(I) ions per protein molecule, an unexpected result for a protein containing two CXXC metalbinding domains. A possible explanation is that one of the domains is especially sensitive to oxidation and thus does not bind copper under the experimental conditions. However, thiol detection indicates that WD12 contains 4.0 Ϯ 0.5 free thiols, suggesting that both domains retain the ability to bind metal. The stoichiometry could also be attributed to the presence of a copper ion already bound in one of the sites, but atomic absorp- tion analysis indicates that WD12 prior to the ITC experiment contains ϳ0.04 copper atoms per protein molecule. Despite the lower quality of the data, the K a value of ϳ10 5 M Ϫ1 indicates that WD12 has a lower affinity for Cu(I) than WD34 and WD56 but approximately the same affinity as Atox1 (Table II).
The WD16 data (Fig. 8) were analyzed with a single site model as well. The stoichiometry of 4.92 Ϯ 0.66 is consistent with the binding of 2 Cu(I) ions to WD34 and WD56 but just one Cu(I) ion to WD12. The K a of ϳ10 5 M Ϫ1 is very similar to that measured for Atox1. In several titrations, including that shown in Fig. 8, slight dips in the curves were evident, sug-gesting that more than one type of site may be present. We tried to account for these dips with a two-site model, but unreasonable stoichiometries (Ͼ10 copper ions) inconsistent with the number of possible ligands were obtained. Fixing the n values did not improve the fits and gave poor agreement among multiple data sets. Therefore, the single site model is most appropriate for WD16 and suggests that the binding sites in WD16 have the same affinity for Cu(I) as one another and as Atox1.
Unlike WD16, the presence of multiple binding sites in WD14 was clear from the titration curve (Fig. 9) and was completely reproducible. When fitted to a one-site model, the stoichiometry was unreasonably low and the fit obviously poor. By contrast, a two-site model gave n 1 ϳ1 and n 2 ϳ3 (Table II). The binding constants have large standard deviations but suggest that the site with n 1 ϳ1 has a higher Cu(I) affinity than the other three sites in WD14. Moreover, this site binds copper more tightly than the sites in all the other WND variants studied (Table II). The class of sites with n 2 ϳ3 is comparable in affinity to WD34 and WD56. Although the stoichiometry is reasonable for a protein containing four CXXC motifs, it is not consistent with the stoichiometries of ϳ1, ϳ2, and ϳ5 measured for WD12, WD34, and WD16, respectively. Based on these values, WD14 might be expected to bind 3 rather than 4 Cu(I) ions.

DISCUSSION
The ITC results presented here provide the first thermodynamic parameters for Cu(I) binding to the Wilson disease protein and its copper chaperone. Values of n, K a , and the apparent ⌬H°have been obtained for Atox1 and five variants of the Wilson protein, WD12, WD34, WD56, WD16, and WD14 (Table  II). The measured stoichiometries are generally correlated with the number of CXXC motifs in each polypeptide. Atox1 binds one Cu(I) ion, indicating that the metal-bridged dimer observed in the crystal structure (15) does not form at low concentrations in solution. The measured n value of 1.41 Ϯ 0.22 could also be construed as binding more than one Cu(I) ion but is most likely due to an error in the protein concentration determination. The stoichiometry of ϳ2 for WD34 and WD56 is consistent with the presence of two CXXC motifs, and the two classes of sites for WD14 have n values of ϳ1 and ϳ3, reflecting the presence of four CXXC motifs. By contrast, WD12 binds only one Cu(I) ion, and WD16 binds closer to 5 Cu(I) ions, consistent with WD12 binding only one Cu(I) ion.
The binding of a single Cu(I) ion by WD12 is unexpected and cannot be explained by oxidation or a tightly bound Cu(I) ion present prior to the ITC experiments. It may be that one of the domains does not bind copper at all. Alternatively, the two domains could coordinate a single Cu(I) ion with the cysteines from two CXXC motifs, analogous to what is observed in the crystal structure of Cu(I)-loaded Atox1 (15). Domains 1 and 2 are separated by a 25 residue linker region, which could allow the two domains to orient properly for this type of coordination. This model could also explain surface plasmon resonance data indicating that MNK domains 1 and 2 have a lower affinity for Atox1 than the other domains (33). Although this finding may be because of the presence of a glutathione S-transferase tag (33), it could also result from the two domains coordinating a single Cu(I) ion. Because the binding surface of the target domain is proposed to involve regions near the CXXC motif (15,37), coordination of a single Cu(I) ion between the CXXC motifs in MNK domains 1 and 2 could hinder Atox1 access.
The K a value of ϳ10 5 M Ϫ1 for Atox1 represents the first report of a Cu(I) binding constant for a copper chaperone. For comparison, the apparent K a for the first Cu(II) ion binding to dimeric copper, zinc superoxide dismutase, is 4 ϫ 10 15 M Ϫ1 at  (38), and the four higher affinity Cu(II) sites in dopamine ␤-monooxygenase have K a values of ϳ10 11 M Ϫ1 (39). Because Atox1 functions to shuttle copper ions within the cell, it is reasonable that it would have a lower affinity for copper than these metalloenzymes. Metallothionein, which binds Cu(I) with sulfur ligands, has a stability constant of 10 17 to 10 19 M Ϫ1 (40). Given the moderate affinity of Atox1 for Cu(I) and the limited free copper concentration (Ͻ10 Ϫ18 M) in the cell (41), how is Atox1 able to retain copper ions until it reaches its target WND domains? One likely possibility is that the kinetic rate constant, k off , is slow, preventing the loss of copper to other higher affinity binding sites. In support of this notion, exchange rates between enzymes and copper are typically slow because of the strength of Cu(I) and Cu(II) binding to ligands (42).
It is not known which WND domains receive copper directly from Atox1, although the strongest interaction by the yeast two-hybrid assay is observed for WD14 (32). If transfer is under thermodynamic control, the WND metal domains, or at least the subset that receives Cu(I) from Atox1, would be expected to have a higher affinity for Cu(I). With the exception of WD14, the K a values for the Wilson domains are all on the order of 10 5 to 10 6 M Ϫ1 . Affinities of this magnitude have been reported previously for the six MNK domains (43) and are consistent with EC 50 values measured for the activation of WND by cop-per (28,44). Importantly, the K a value for WD16, the most physiologically relevant variant, is essentially the same as that for Atox1 (Table II). The chaperone and target domains thus have similar affinities for Cu(I). This result is consistent with an exchange constant of ϳ1.4 measured for copper transfer between the yeast chaperone Atx1 and one domain of its target ATPase, Ccc2 (45). If copper delivery from Atox1 to the WND N-terminal domains is followed by rapid transfer to another site in the ATPase, the ultimate driving force could derive from ATP hydrolysis. Because copper transfer between Atox1 and the WND metal-binding domains is not governed by a thermodynamic gradient, it must be under kinetic control. As suggested above, Atox1 may have a slow k off for copper, allowing it to function in the copper-limiting environment of the cytoplasm. Complexation between Atox1 and WND could alter k off and lower the kinetic barrier for transfer between metal-binding sites as suggested previously for Atx1 and Ccc2 (45). Similarly, interactions between Atox1 and its copper donor, which could be the membrane transporter Ctr1 (46) or another yet to be identified factor (47), could modulate k on .
Despite the overall similarity in binding constants for Atox1 and the WND domains, some differences between the WND domains are apparent from the data. An approximate hierarchy of binding affinities is WD14 2 Ͼ WD34 Ն WD56 Ϸ The values for ⌬H°are apparent and include contributions not only from Cu(I) binding but from associated events such as deprotonation of the two cysteines in the CXXC motif and changes in the buffer ionization state.
c The WD14 data were best fit with a two-site model.
. This variability in binding constants for the WND proteins has important implications. Both WD34 and WD56 bind copper more tightly than WD12 and Atox1. This distinction is not apparent for the intact WD16, however. Furthermore, data for WD14 are best fitted with two distinct types of sites with one binding constant that is higher than those observed for WD16 or any of the other variants. This finding is incompatible with the one-site, lower affinity K a value obtained for WD16. The copper affinities of the WND CXXC-containing domains are therefore somewhat different in the presence of the other domains. A potential explanation for the strikingly different results obtained for WD14 versus WD16 is that the WD14 protein adopts a different conformation than the intact WD16. A different -fold could also explain why WD14 gives approximately five times more signal in a yeast two-hybrid assay than WD16 (32). Of all the intervening sequences connecting the six metal-binding domains, the linker between domains 4 and 5 is the longest, consisting of 69 residues, and could cause tertiary structural differences between WD14 and WD16. Many previous biochemical, biophysical, and genetic studies of the WND and MNK metalbinding domains have focused on proteins comprising fewer than six domains. Our results indicate that the sum of the parts is not the same as the intact N terminus and underscore the importance of utilizing all six domains in future work.
Based on the ITC data, the discrete functions of the WND metal-binding domains are not defined by copper-binding affinities. How then are the domains distinguished from one another? Consideration of the electrostatic properties of each domain furnishes part of the answer. Interactions between Atox1 or Atx1 and their target domains involve complementary electrostatic surfaces (15,16,37,48). In particular, positively charged residues on the chaperone surface interact with negatively charged residues on the target domain. For the yeast system, the predicted isoelectric points of Atx1 and the two N-terminal domains of Ccc2 are 8.6, 4.3 and 4.0, respectively, suggesting that Atx1 might interact similarly with each Ccc2 domain. The situation is more complicated for Atox1 and WND. The isoelectric point of Atox1 is 6.7, whereas the isoelectric points for the six WND domains range from 3.8 to 8.7 (49). It seems unlikely that protein-protein interactions between Atox1 and each of the six different domains could facilitate copper transfer in the same way. Instead, only some of the domains might exchange copper with Atox1. Yeast two-hybrid data indicate that a polypeptide comprising domains 5 and 6, which are proposed to function in copper translocation (28), does not interact with Atox1 (32,33). Domains 5 and 6 could receive copper from domains 1-4, however. Alternatively, in the intact WD16, domains 5 and 6 might interact directly with Atox1. As noted above, the presence of the other domains can affect the behavior of the individual domains, and data acquired on truncated proteins should be interpreted carefully. Domains 1-4 have been suggested to function in copper-responsive localization (27,28). Their electrostatic properties and/or overall structure could confer such a function by modulating interactions with additional regulatory proteins. A possible candidate is the Murr1 protein. This protein, proposed to be involved in hepatic copper toxicosis, has recently been shown to interact directly with the six WND metal-binding domains (50).
In sum, ITC has been used to measure values of n and K a for Cu(I) binding to the copper chaperone Atox1 and various combinations of the metal-binding domains of its target transport ATPase WND. The association constants are ϳ10 5 to 10 6 M Ϫ1 and are similar for chaperone and target domains. These data suggest that copper handling by Atox1 and copper exchange between Atox1 and WND are under kinetic rather than thermodynamic control. Some differences in both n and K a values are observed for proteins containing less than the full complement of six metal-binding domains, demonstrating that the copper-binding properties of each domain are affected by the presence of the other domains. The data for WD16, however, were best fitted with ϳ5 Cu(I) sites all with the same binding constant. Consequently, the functions of the six WND metalbinding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties. Detailed characterization of the interactions between WD16 and its partner proteins is therefore critical to delineating the roles of the individual domains.