From the
Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921 and the
Department of Biochemistry and Molecular Biology, School of Medicine, Oregon Health and Science University, Portland, Oregon 97239
Received for publication, April 3, 2003
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ABSTRACT |
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INTRODUCTION |
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Copper transport to membrane-bound proteins such as copper-transporting ATPases or delivery to copper-dependent enzymes is carried out by small cytosolic proteins called chaperones that are specific to each protein. HAH1 (3, 4) is the chaperone (5) for Menkes (MNK)1 and Wilson's disease proteins (WND) (611). These copper-transporting ATPases are located in the trans-Golgi apparatus and are believed to provide copper for ceruloplasmin (12), peptidylglycine monooxygenase (13), and tyrosinase (14). They share 50% sequence homology and differ mainly in their location in the body. Whereas WND is predominantly found in the liver and brain tissues, MNK is found in all other tissues but liver and brain. HAH1's counterpart in yeast, Atx1 (15, 16), delivers copper to Ccc2, the yeast homologue for MNK and WND (17). COX17, COX11, and SCO1 are involved in copper shuttling to the mitochondrial membrane and incorporation of copper into cytochrome c oxidase (1821). Copper chaperone for superoxide dismutase shuttles copper to Cu,Zn-superoxide dismutase (2227).
Solution structures for several of the copper-bound and apo forms of chaperones as well as isolated metal-binding repeats of some ATPases have been published (2836). All structures adopt a fold. The conserved metal-binding motif, MX-CXXC, which is anticipated to bind Cu(I), is located in the connecting loop between the first
-sheet and the first
-helix. A Hg(II) derivative of the Atx1 chaperone from Saccharomyces cerevisiae was characterized by crystallography to reveal a linear two-coordinate Hg(II) center ligated to Cys15 and Cys18 (30). NMR studies on Cu(I)-loaded Atx1 and Bacillus subtilis CopZ revealed a slightly different environment in the vicinity of the bound Cu(I), more indicative of a three-coordinate structure (28, 29), with SCuS angles of 120 ± 40° and 115 ± 26°, respectively. Comparison with the NMR-derived apo structures (28, 29) indicated that one of the ligated cysteines (the one closest to the N terminus) occupies a different conformation in the apo proteins, where it points away from the metal-binding pocket toward solvent. This residue appears to flip inwards as the result of Cu(I) binding becoming much less conformationally mobile in the process. Similar conclusions have been obtained from the NMR structures of apo and Cu(I)-loaded forms of the chaperone ATPase partners S. cerevisiae Ccc2a (35) and B. subtilis CopAb (36) (where "a" and "b" denote the first and second subdomains, respectively). These proteins likewise show a large conformational change in the position of the N-terminal cysteine residue as it flips from a solvent-exposed conformationally mobile location in the apo form to its Cu(I)-coordinated position. Again, the SCuS angles are estimated to be 115° and 132°, respectively, much less than the 180° value expected for linear two-coordination. Three-coordination involving an exogenous thiol has been proposed as the origin of the third ligand, since all potential protein-derived third ligands are at too great a distance to influence the Cu(I) coordination directly.
NMR spectroscopy does not directly observe the bound copper ion or the putative exogenous ligand (37). Assumptions about the copper coordination are made on the basis of the known van der Waals radius of the Cu(I) ion and the distance of the coordinating atoms (i.e. two cysteine-sulfurs in these chaperones). X-ray absorption spectroscopy (XAS), on the other hand, is unique in its ability to define the local coordinate structure of the otherwise spectroscopically silent Cu(I) ion in proteins and reveals detailed information about the immediate coordination environment within a radius of <5 Å. Metal-ligand distances can be determined to high accuracies (0.02 Å), whereas information on coordination number and oxidation state of the metal can also be determined. XAS spectroscopy at the copper K-edge has been used to confirm the presence of two-coordinate Cu(I) in MNK (38) and WND (39), where the Cu(I) center is ligated by the two cysteine residues of the MXCXXC motif. However, XAS studies by Penner-Hahn and co-workers (40) on the copper-bound form of Atx1 in S. cerevisiae found a three-coordinate Cu(I) center bound by two "short" (2.24-Å) and one "long" (2.4-Å) CuS interaction. As with the NMR studies, it was suggested that the third ligand was derived from the reductant, dithiothreitol (DTT), that had been added to the protein prior to metal reconstitution. George and co-workers (41) have performed EXAFS spectroscopy on Enterococcus hirae CopZ and the full-length and second metal-binding motif of MNK (42). Again, rather than the expected digonal coordination, mixtures of two- and three-coordinate species were found. Most recently, an XAS study of B. subtilis Cu(I)-CopZ by Banci et al. (37) has reported a three-coordinate environment (three CuS at 2.25 Å) in both DTT- and dithionite-reduced samples and in the presence of carboxylate anions as potential exogenous ligands. Ascorbate reduction, on the other hand, was found to elicit a perturbed spectrum with evidence for a low Z scatterer (CuO at 1.95 Å) in addition to the two S scatterers (two CuS at 2.25 Å).
These studies pose two important questions. First, can the two-coordinate Cu(I)-bound chaperone be prepared free from ligation by an exogenous third ligand, and if so, what is the structure? Second, what is the role (if any) of binding of a third ligand in chaperone function? In the present study, we report the results of biochemical and XAS studies of the HAH1 metallochaperone, which for the first time reveal the presence of a linear two-coordinate Cu(I) center, free from ligation by any exogenous ligand. We have also investigated the affinity of this two-coordinate site for the exogenous thiol ligands, GSH and DTT, as well as for the phosphine ligand tris-(2-carboxyethyl)-phosphine (TCEP), which is often used as a reductant for disulfides in proteins. Our results establish that exogenous ligands can bind to the two-coordinate Cu(I) center but with rather low affinity.
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EXPERIMENTAL PROCEDURES |
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Protein masses were determined by electrospray ionization mass spectrometry. Proteins were injected onto a 1.0 x 250-mm C4 column (214 MS C4; Vydac), and masses were determined on-line using a model LCQ ion trap (ThermoFinnigan). The flow rate was 20 ml/min with a linear gradient of 1850% acetonitrile over 40 min in a mobile phase containing 0.2% acetic acid and 0.005% heptafluorobutyric acid. Samples were autoinjected and concentrated/purified using a microprotein trap cartridge (Michrom Bioresources, Inc.). Mass spectra of proteins eluted from the C4 column were deconvoluted using BioWorks Browser software (ThermoFinnigan). Mass accuracy of better than 0.01% was confirmed using horse myoglobin.
Metal ReconstitutionAll procedures described below were performed in an inert atmosphere in an anaerobic chamber to prevent oxidation of the cysteine residues. Copper was added as the tetraacetonitrile Cu(I) complex to minimize disproportionation of free Cu(I) to Cu(II) and metallic copper. DTT (Sigma) was used to reduce the cysteine residues in HAH1 prior to metal reconstitution. DTT was added as a 10-fold excess to the protein, and the mixture was incubated for 10 min on ice. The protein was then dialyzed overnight under argon into a buffer containing 50 mM HEPES (Sigma) and 10% acetonitrile (Sigma) at pH 7.5. Copper was added in a 1:1 ratio by slow infusion of a 100-fold stock solution of [(CH3CN)4Cu(I)]PF6 over a 1-h period utilizing a syringe pump at a rate of 1 µl/min. Successive dialyses against HEPES plus 10% acetonitrile, HEPES plus 5% acetonitrile, and HEPES without acetonitrile for 12 h per buffer in a Slide-a-lyzer cassette (Pierce) yielded reconstituted CuHAH1. In all samples, copper/protein ratios were between 0.4 and 1.0 as determined by flame atomic absorption spectroscopy (Varian AA-5). The copper content of the sample was monitored throughout the various dialysis steps. In a typical experiment, 8 ml of 100 µM protein was reconstituted to a final copper concentration of
5080 µM. The sample was sealed and stored at80 °C. For XAS experiments, the protein was concentrated using an Ultrafree centrifugation system (Millipore Corp.; molecular mass cut-off, 3500 Da) modified to maintain an anaerobic atmosphere. In this protocol, a concentrator was filled with dilute sample and sealed with a rubber septum in the anaerobic chamber and then transferred to a tabletop centrifuge for concentration outside the chamber. The oxidation state of copper in the final sample was checked by EPR (Bruker Elexsys). The Cu(I)/Cu(II) ratio was determined by comparison of the integral of a cavity/base line-corrected EPR spectrum of Cu(I)HAH1 to a 100 µM Cu(II) EDTA sample. Concentrations of Cu(II) never exceeded 10% and were typically around 24%.
Titrations with Exogenous LigandsBinding of three commonly used reductants (GSH (Sigma), DTT (Sigma), and TCEP (Sigma)) to CuHAH1 was probed in two sets of experiments. All three substances provide a potential ligand to CuHAH1, either a sulfhydryl group (DTT and GSH) or a phosphine group (TCEP). In the first set of experiments, GSH, DTT, and TCEP were added in a 10-fold excess (typically 10001500 µM) to the apoprotein and incubated for 1 h before adding copper. The sample was then reconstituted with copper and dialyzed as described above.
In the second set of experiments, the three ligands were each titrated into the protein after metal reconstitution, with ratios of 1:1, 5:1, and 25:1 reductant/protein. After the addition of the ligand, the samples were concentrated to 100 µM [Cu] for XAS measurements. For a complete list and description of all XAS samples, see Table I.
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XAS Data Collection and AnalysisXAS data were collected in March, May, and December of 2002 on beam line 9-3 at the Stanford Synchrotron Radiation Laboratory. Beam line 9-3 was operated at 3.0 GeV with beam currents between 50 and 100 mA. The samples were measured in fluorescence mode using a 30-element array detector (Canberra). A rhodium-coated mirror positioned upstream of the Si220 monochromator was used to cut off all energy above 12 keV. A second mirror located downstream from the monochromator was used to focus the beam (5 x 1 mm). Soller slits with a 6-µ nickel filter were placed in front of the detector to selectively attenuate the elastic scatter peak with respect to the copper K fluorescence. Under these conditions, no dead time corrections were necessary. The samples were measured at 1015 K using a liquid helium flow cryostat. Data for each channel were inspected for background noise and glitches. The nickel K
fluorescence, which is generated by the nickel filter and is always present in the single channel analyzer window, was removed by collecting data on a blank sample under identical conditions of detector/Soller slit geometry and subtracting this as a background file from each data set. The energy was calibrated by assigning the first inflection point of the copper edge from a copper metal foil that was inserted between the second and third ionization chamber to 8980.3 eV. The number of scans collected for each sample varied from 2 to 15, depending on the copper concentration of the samples. The scans were collected to k = 12.8 Å1 because of interference by traces of zinc in the sample.
Data reduction and analysis were performed using the EXAFSPAK computer suite (43). Theoretical phase and amplitude functions were calculated using FEFF 8.0 (44). The inspected raw data were averaged, background-subtracted, and normalized. The energy corresponding to the start of the EXAFS (k = 0) was set at 9000 eV. A k4-weighed spline fit was used to extract the EXAFS oscillations from the raw data. The EXAFS data were simulated by curve-fitting in the OPT module of EXAFSPAK using a nonlinear Marquadt algorithm. The following parameters were refined: E0 (a small energy correction at k = 0), Ri (the distance between the central absorber and atom i), and
2 (the Debye-Waller factor, defining the mean square deviation of Ri). A goodness of fit (F) parameter was used to evaluate the merit of the fit. F is defined as follows.
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RESULTS |
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Metal reconstitution of HAH1 with Cu(I) poses two problems. The metal binding cysteines in the CXXC motif of HAH1 are exposed to solvent and therefore prone to oxidation and formation of a disulfide bridge (28, 29, 45) (Fig. 1c). To prevent oxidation within the unoccupied metal binding site, experiments have to either be performed anaerobically or in the presence of a reductant. In addition, Cu(I) as the free ion is unstable in aqueous solution and is sensitive to oxidation or disproportionation. Because of the high hydration energy of Cu2+ versus Cu+ (Cu+ = 0.139, Cu2+ = 0.522 kcal/mol), the latter will disproportionate in water into Cu2+ and Cu0. However, complexes like [Cu(I)CN4]3 or [Cu(I)(CH3CN)4]+ stabilize Cu(I), whereas maintaining dilute Cu(I) concentrations will shift the disproportionation equilibrium toward the Cu(I) species.
To address both problems, we performed all experiments in an anaerobic chamber and used [Cu(I)(CH3CN)4]PF6 with 10% acetonitrile (the complexing ligand) present in all buffers during reconstitution. The copper was added through a syringe pump over a period of 1 h. During the addition, the sample was gently agitated to prevent the accumulation of high local Cu(I) concentrations. After the addition, the amount of acetonitrile in the buffer was reduced stepwise by 5% to 0% by dialysis. Storage of concentrated samples at80 °C instead of 22 °C turned out to be crucial to the integrity of the samples. No precipitation or oxidation was observed after storage times of over 2 months. Earlier experiments had shown that even after insertion of copper into the protein, exposure to oxygen leads to at least partial oxidation and loss of Cu(I) from its binding site. EPR spectra of samples that were dialyzed and concentrated under aerobic conditions showed that after 48 h, 2530% of the copper had been oxidized to Cu(II) (data not shown).
Table I lists the identity of the samples investigated by XAS, their copper concentrations, and their copper/protein ratio. Three series of experiments were carried out: (i) HAH1 was reduced with DTT and dialyzed to remove the thiol prior to addition of copper; (ii) a 10-fold excess of reductant (GSH, DTT, and TCEP) was added immediately prior to the addition of copper, followed by successive dialysis steps to remove acetonitrile and unbound metal; and (iii) reductant (GSH, DTT, and TCEP) was titrated into the copper-reconstituted sample. The second and third experiments differ in that excess thiol is removed by dialysis in the second, whereas it remains available to complex the protein-bound copper in the third. The EXAFS spectra for all samples were dominated by a strong CuS back-scattering. For CuHAH1 in the absence of any reductant, two samples from two independent preparations were analyzed to ensure reproducibility of the results (HAH11 and HAH12); parameters used to fit the data are shown in Table II. The EXAFS data exhibited one main frequency (Fig. 2, inset), which was also reflected in the Fourier transform (FT) with one main peak centered at 2 Å and some minor peaks between 3 and 6 Å (Fig. 2). An excellent fit could be obtained with two CuS at 2.16 ± 1 Å, a distance typical of linear SCuS coordination (46, 47). No significant improvements to the fit were achieved by including a second shell in the model.
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More information about the copper coordination in a complex can be extracted from the x-ray absorption near edge structure (XANES) region, which spans the range 10 eV below to 10 eV above the absorption edge, and provides information about electron transitions and the oxidation state of the absorbing atom. XANES spectra of Cu(I) complexes show a pre-edge feature at 8983 eV, which is due to a 1s
4px,y electronic transition, the shape and intensity of which depends on the local copper coordination geometry (46, 48, 49). The transition shows the highest intensity and resolution for linear two-coordinate complexes and becomes less intense and broadened as the coordination increases and/or the site symmetry is lowered. Thus, it can be used as an indicator of two-versus three-coordination. As expected for a linear coordinated Cu(I) ion, the XANES spectrum for HAH1 showed an intense pre-edge feature (Fig. 3). The intensity of the pre-edge feature of Cu(I) HAH1 was almost identical to that of the linear two-coordinate model compound bis-2,3,5,6-tetramethylbenzenethiolate Cu(I) (47), suggesting a linear SCuS bond angle close to 180° in the Cu(I) chaperone. We conclude that the copper in HAH1 is in a digonal bis-cysteinate environment. In contrast to the NMR studies and the earlier XAS studies on Atx1, the data showed no indication of the presence of a third ligand.
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The previous proposal, that commonly used reductants such as DTT or GSH could act as a potential source of additional ligation, led us to the experiments in which we reconstituted HAH1 in the presence of a 10-fold excess of either DTT (HAH1-DTT), GSH (HAH1-GSH), or TCEP (HAH1-TCEP) (Table I). Fig. 4, a and b, shows a comparison of the FTs and the EXAFS for the three reductants, respectively. All three samples were comparable in copper/protein ratios (0.50.6) and in total copper concentrations (250400 µM). The EXAFS data of HAH1-DTT and HAH1-GSH showed the same digonal, almost linear SCuS environment. CuS distances for both samples were refined to 2.16 ± 1 Å (HAH1-GSH) and 2.17 ± 1 Å (HAH1-DTT), respectively, with an F parameter of 0.30 (HAH1-GSH) and 0.29 (HAH1-DTT). No indication of a third ligand was found. Accordingly, the FTs of HAH1-GSH and HAH1-DTT looked almost identical to HAH11 and HAH12, with the former spectra exhibiting a better signal/noise ratio. Comparison of the intensity of the 8983 eV edge feature for the samples is shown in Fig. 4c. It is obvious that adding a 10-fold excess of DTT or GSH to HAH1 prior to metal reconstitution has no effect on the copper coordination in the protein. On the other hand, the 8983-eV edge feature for HAH1-TCEP is much less intense and slightly shifted toward lower energy and is characteristic of a three-coordinate Cu(I) complex (46). As in HAH12, the EXAFS data of HAH1-TCEP is dominated by strong CuS/P backscattering, but the intensity of the oscillations and of the main peak in the FT is greater than for the former. The EXAFS data were best fit with three CuS at 2.24 ± 1 Å. We assume that the actual coordination is two CuS plus one CuP as the third ligand and is most likely provided by a phosphine group from TCEP. EXAFS spectroscopy cannot distinguish between elements that are next to each other in the periodic table.
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We conclude that metal reconstitution of HAH1 in the presence of a 10-fold excess of DTT or GSH to prevent local oxidation of the cysteinates has no impact on the copper coordination. However, use of TCEP as reductant results in binding of the phosphine as an exogenous ligand to Cu(I). This implies a lower affinity of Cu(I)HAH1 for SH groups of GSH and DTT than for the PR3 groups of TCEP.
To further probe the binding of a third ligand, we titrated reconstituted Cu(I)HAH1 with specific ratios of DTT, GSH, and TCEP (1:1, 5:1, and 25:1, ligand to copper, respectively). The results of the best fits to the EXAFS are listed in Table II. Adding equimolar amounts of DTT or GSH to CuHAH1 has no impact on the resulting copper coordination (Fig. 5, a and b). Increasing the amount of reductants to 5-fold does not change the coordination (data not shown). However, the spectra for a 25-fold excess of thiol ligand show an altered copper coordination. In HAH1-DTT25, the CuS distance is elongated to 2.20 ± 1 Å with a coordination number of 2. For HAH1-GSH25, the data best fit 2.5 CuS at 2.22 ± 1 Å. Fig. 6, a and b, summarizes the results for the TCEP titration of CuHAH1. When added as equimolar amount to CuHAH1, a linear CuS coordination was observed with two CuS at 2.14 ± 1 Å. However, adding a 5-fold excess of TCEP to CuHAH1 shows an already completed conversion to three-coordination, which confirms the higher affinity of CuHAH1 for phosphine over thiol. Fitting the EXAFS for HAH1-TCEP5 and HAH1-TCEP25 gave identical results of three CuS at 2.24 ± 1 Å. The changes in coordination with higher ligand/copper ratios for this experimental series are also reflected in a lowered intensity of the 8983-eV edge feature, which is depicted in Fig. 7.
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Coordination numbers in EXAFS spectroscopy are not well defined because of their high correlation with the Debye-Waller factor. Thus, errors of ±30% are not uncommon. However, distances derived from EXAFS data are defined to high accuracy (±0.01 Å or better) and can be used to quantitatively determine the ratios of two- and three-coordinate CuS species within a sample (46). Using the equation of Pickering et al. (46) with pure two- and three-coordinate CuS distances of 2.16 and 2.25 Å, respectively, the fractions of two- and three-coordinate copper species in our samples can be estimated. For DTT, equimolar and 5-fold excess of ligand show no increase in CuS above the two-coordinate value of 2.16 Å, whereas a 25-fold excess gives an average CuS distance of 2.20 Å, corresponding to 65 ± 10% of the copper remaining in a two-coordinate site and 35 ± 15% of the copper in a three-coordinate site. For GSH, we calculate trigonal fractions of 25 ± 10, 16 ± 9, and 43 ± 12%, for 1:1, 5:1, and 25:1 ratios, respectively. For TCEP, the trigonal fractions are 0 ± 8, 84 ± 16, and 84 ± 16%, respectively. The error of these ratios is calculated, considering an inaccuracy of the refined distance of 0.01 Å. It should be noted that the calculated ratios for TCEP are likely to be less precise, because the copper coordination consists of two S and one P ligand, which alters the base distances used in our previous calculation.
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DISCUSSION |
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The method of reconstitution was critical to the preparation of the unligated two-coordinated Cu(I) chaperone species. Although we used DTT and/or GSH as a reductant of disulfides prior to copper reconstitution, we were successful in preparing Cu(I) chaperone complexes free from exogenous third ligand coordination. The key to success was to ensure the presence of a high concentration of a weakly coordinating ligand, which can compete effectively with thiols during the reconstitution process. Thus, use of [(CH3)CN)4Cu]+ as the source of Cu(I), with a large excess of acetonitrile (10%) maintained in the buffer during initial copper loading, resulted in good levels of reconstitution (0.51.0 molar equivalents). It is likely that the excess acetonitrile served to completely sequester the Cu(I), rendering it soluble and stable to disproportionation during subsequent dialysis. It is also possible that thiols were more effectively removed during dialysis as mixed Cu(I)-acetonitrile-thiol complexes. Whatever the mechanism, XAS measurements showed that the Cu(I) centers are linear two-coordinate with CuS bond lengths of 2.16 Å, a value typical of digonal copper (38, 46, 47). Additionally, the x-ray absorption edge exhibited an intense feature at 8983 eV of the same magnitude as that of the rigorously linear two-coordinate bis-2,3,5,6-tetramethylbenzenethiolate Cu(I) complex, which has CuS distances of 2.137 Å and a SCuS angle of 178.6° (47). Taken together, these parameters unequivocally establish the linear two-coordinate geometry of the unligated form of the Cu(I)HAH1 chaperone.
With a genuine two-coordinate Cu(I) chaperone species in hand, we next investigated whether exogenous ligands could bind to generate the kind of three-coordinate species postulated from NMR. The addition of the thiol-containing ligands GSH and DTT had little or no effect on the XAS spectra at 1:1 ratios, but increasing concentrations of the thiol led to a decrease in intensity of the 8983 eV peak and a gradual increase in the average CuS bond length (Table II). Pickering et al. (46) have carried out systematic studies of Cu(I)-thiolate clusters and have established that the mean CuS distance determined by EXAFS increases monotonically with the fraction of trigonal to digonal copper. Although titration with GSH and DTT clearly decreased the fraction of two-coordinate Cu(I) in favor of a three-coordinate component, this process was not complete even at a 25-fold molar excess of ligand (7 mM GSH, 25 mM DTT). Using the empirical relationship derived by Pickering et al. (46), we estimate 43 and 35% three-coordinate contribution for GSH and DTT, respectively. Since the KD for binding of the third ligand equals the concentration of ligand required to induce 50% conversion to the three-coordinate species, this puts a lower limit of 1025 mM for the KD for thiol binding. Of further significance, use of DTT or GSH as reductants prior to the Cu(I)-acetonitrile reconstitution protocol did not lead to exogenous thiol coordination.
Titrations with the phosphine TCEP showed evidence of a higher affinity for third ligand binding. Although a 1:1 molar ratio of phosphine to copper had little effect on the spectroscopy, the titration appeared complete at a 5:1 molar ratio (5 mM TCEP), implying a KD between 1 and 5 mM. Furthermore, samples reconstituted in the presence of phosphine showed three-coordination even after exhaustive dialysis. TCEP thus appears to bind more strongly to the Cu(I) centers in chaperones to produce a complex that is resistant to removal by dialysis and hence must be rather inert kinetically.
It is unclear whether the estimated magnitude of KD for thiol ligation to HAH1 is small enough to explain the presence of a third ligand in the NMR and EXAFS studies from other laboratories. Cobine et al. (41) rigorously excluded thiols from the preparation of their sample for XAS yet still observed a three-coordinate site. The authors suggest that the chloride ion, which has phase and amplitude parameters similar to sulfur, might be the origin of the third ligand. The NMR studies of Banci and co-workers (28, 29) on CopZ and Atx1 were prepared by methods similar to the initial EXAFS measurements of Pufahl et al. (40) on Atx1, utilizing a 20-fold excess of DTT for reduction but following this by an ultrafiltration or dialysis step. This experiment was similar to that carried out in the present study, which yielded a two-coordinate Cu(I) site with no bound third ligand.
Another potential source of a third ligand is dimerization. A crystal structure of HAH1 has shown the feasibility of dimerization mediated by metal binding, with Cu(I) strongly coordinated by two cysteines of one monomer and one cysteine of the other. The fourth cysteine appears weakly bound in pseudotetrahedral geometry (31). Kihlken et al. (50) have recently reported that Cu(I) binding induces dimerization of B. subtilis CopZ but only in the absence of DTT. Presumably, binding of DTT to the bis-cysteinate Cu(I) center prevents dimerization (28, 29). The recent XAS and NMR studies of the B. subtilis CopZ suggest that nonspecific protein aggregation may occur at high protein concentrations (37). If dimerization or aggregation is a contributing factor to the observation by other laboratories of three-coordinate structures, it is possible that the conditions used to reconstitute HAH1 in this study favored the monomeric state. Notwithstanding these uncertainties, we have firmly established the existence of the two-coordinate form of the HAH1 chaperone and its ability to form three-coordinate adducts with thiol ligands, albeit with low affinity. These results provide evidence in support of proposed mechanisms (31, 34, 36) for copper transfer in which the solvent-exposed unligated N-terminal cysteine of the apo-partner flips inward to interact with the two-coordinate Cu(I) center of the chaperone and form a three-coordinate intermediate within a chaperone-partner heterodimer. The relatively low affinity of the exogenous thiol interaction with the two-coordinate Cu(I) center suggests that it would be insufficient to drive the heterodimer formation on its own so that electrostatic complementarity (predicted from NMR structures) between chaperone and ATPase domain surfaces would provide additional driving force for metal transfer (34, 36). The interplay between these two factors most likely contributes to the selectivity of the chaperone for individual subdomains of the ATPase partner (5) and to the observed reversibility of copper transfer from ATPase to chaperone, which has been proposed as an important regulatory mechanism of copper homeostasis (51). With respect to the latter, we note that the reverse mechanism would require the two-coordinate center on the ATPase (38, 39) to also be able to form adducts with exogenous thiol ligands to generate the common intermediate. Studies aimed at establishing this chemistry are under way.
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FOOTNOTES |
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¶ To whom correspondence and reprint requests should be addressed: Dept. of Environmental and Biomolecular Systems, OGI School of Science & Engineering, 20000 N. W. Walker Rd., Beaverton, OR 97006-8921. Tel.: 503-748-1384; Fax: 503-748-1464; E-mail: ninian{at}bmb.ogi.edu.
1 The abbreviations used are: MNK, Menkes disease protein; DTT, dithiothreitol; EXAFS, extended x-ray absorption fine structure; FT, Fourier transform; TCEP, tris-(2-carboxyethyl)-phosphine; WND, Wilson's disease protein; XANES, x-ray absorption near edge structure; XAS, x-ray absorption spectroscopy.
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ACKNOWLEDGMENTS |
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REFERENCES |
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