From the
Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, Vic 3125, Australia,
ZMBH Centre for Molecular Biology, University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany, and the ||German Cancer Research Center, Im Neuenheimer Feld 506, Heidelberg D-69120, Germany
Received for publication, December 6, 2002 Accepted for publication April 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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In yeast, Atx1 was localized to the cytosol, and its role in the copper trafficking pathway and in iron metabolism was described (22). A trigonal coordination chemistry between copper and Atx1 was proposed (23), and a copper-dependent interaction of the N terminus of the yeast MNK homologue Ccc2 and Atx1 was demonstrated by yeast two-hybrid analysis (23, 24). Complex formation and dissociation of Atx1 with Ccc2 was investigated through 1H and 15N chemical shift perturbation experiments and found to be in the order of milliseconds (25). A lysine-rich region resulting in a positively charged face of Atx1 was crucial for intracellular copper trafficking, suggesting an important interaction site between Atx1 and Ccc2 (24).
The functional significance of the six MBSs in WND and MNK is unclear, but there is growing evidence that there is a functional distinction between the sites. The prokaryotic and lower eukaryotic orthologues have only 13 MBSs, suggesting that all six are not required for copper transport. Work from our laboratory has demonstrated that only one of MBS5 or MBS6 of MNK was necessary and sufficient for the protein to traffic to the plasma membrane in response to copper (26). This result showed that MBS5 and MBS6 have some specific function in the copper-induced trafficking process and suggested that the first four metal binding sites may have a separate function. The effect of mutating various combinations of MBSs on the ability of MNK and WND to deliver copper to the ceruloplasmin orthologue, Fet3p, in yeast has been assessed by two groups with markedly different results for the two proteins (27). Payne et al. (27) found that progressive mutations of the MBSs from CXXC to SXXS from the N-terminal end caused a complete loss of copper delivery to Fet3p after only the first two MBSs had been mutated. A zipper-like mechanism for copper binding to MBS14 would explain why Forbes et al. (28) found that only MBS6 of WND was necessary for complete restoration of Fet3p activity. It also was proposed that these results may indicate that the various MBSs perform distinct functions in MNK as compared with WND.
Using both yeast and mammalian cell assays, a copper-dependent interaction of Atox1 with the MBSs in the WND N terminus has been demonstrated (29). Two cysteines in the CXXC motif of Atox1 were necessary for an interaction with WND and a single MBS of WND was sufficient to promote this interaction. Interestingly, MBS5 and MBS6 of WND failed to interact with Atox1. In the same report, an interaction of the complete MNK N terminus with Atox1 was demonstrated, although a functional difference between individual MBSs of MNK had not been investigated.
Yeast two-hybrid analysis is based on an interaction-dependent reconstitution of a transcription factor in the yeast nucleus and hence is an indirect method to measure protein-protein interactions. Yeast two-hybrid screens are subject to artifacts and may miss many interactions or produce false positive results. Therefore, a recently established real-time metallochaperone-target interaction method (30) was applied here, and the data were compared with yeast two-hybrid analysis. As demonstrated for the MNK homologue WND (29), using the yeast two-hybrid assay, we found a copper-dependent interaction of Atox1 with the first four MBSs of MNK but not with MBS5 and MBS6, suggesting a preferential binding of the chaperone with the N-terminal MBSs. However, using surface plasmon resonance (SPR), we detected a copper-dependent transient interaction of the chaperone with all six MBSs. We quantitatively assessed association and dissociation rates and calculated binding constants of 119 x 106 M for all pairs of MBSs with Atox1. Mutations in the MBSs did not significantly affect the overall binding affinity, suggesting that the binding of multiple chaperones to the N terminus of MNK is of a non-cooperative nature.
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EXPERIMENTAL PROCEDURES |
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Yeast Strain, Transformations, and Two-hybrid AssayThe yeast reporter strain L40 (MAT his3
200 trp1901 leu23112 ade2 LYS2::(lexA-HIS3) (URA3::lexA-lacZ) GAL4) (31) was co-transformed with the indicated control plasmids pGAD EEA1wt and pLex EEAwt (33) or pCMB152 (encoding Atox1-Gal4) and various pCMB plasmids using a lithium acetate-based method (34). The co-transformants were selected on complete synthetic medium lacking tryptophan and leucine (Bio 101, La Jolla, CA). The yeast two-hybrid experiments were essentially performed as described in the manual provided by the supplier of the two-hybrid system (Clontech).
Western BlotsProtein extracts from transformed yeast L40 cells were prepared by the trichloroacetic acid method as described in the yeast protocols handbook provided by Clontech. 30 µg of total protein extract were fractionated by SDS-PAGE and transferred onto nitrocellulose (Schleicher & Schuell). Atox1-Gal4 (see Fig. 2A) and MBS-LexA (see Fig. 2B) were detected using monoclonal anti-Gal4 and anti-LexA antibodies, respectively (both purchased from Clontech), followed by a secondary horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amrad Biotech). The detection of peroxidase activity was carried out using the chemiluminescent peroxidase substrate (Roche Molecular Biochemicals). All the protocols were performed essentially as described previously (35).
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Atox1 Peptide SynthesisFor solid phase synthesis of the 69 amino acids containing Atox1, we employed the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy (36, 37) in a fully automated synthesizer (Applied Biosystems 433). Peptide chain assembly was performed using in situ activation of amino acid building blocks by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The purified material was analyzed by high pressure liquid chromatography and laser desorption mass spectrometry (Vision 2000, Finnigan MAT). Purified peptides were dissolved in double distilled water at a concentration of 700 µM and stored at 70 °C until use.
Construction of Vectors for Overexpression of MBSspGEX-4T-2 was obtained from Amersham Biosciences. A 0.7-kb fragment encoding MBS1/2 was amplified from pCMB226 using the primers: MBS1/2fw, 5'-TATAAGGATCCGGTGTGAATTCTGTTACC-3'; and MBS1/2rev, 5'-AATTATGTCGACTTACTTTTTGACAAATGC-3'. A 0.53-kb fragment encoding MBS3/4, MBS3/4-m3, or MBS3/4-m34 was amplified from pCMB222 using the primers: MBS3/4fw, 5'-TATAAGGATCCAATGATTCAACAGCCAC-3'; and MBS3/4rev, 5'-AATTATGTCGACTTAGTCTGACAAGGTAGCATC-3'. A 0.46-kb fragment encoding MBS5/6 was amplified from pCMB225 using the primers: MBS5/6fw, 5'-TATAAGGATCCGGTGTGAATTCATCTAAG-3'; and MBS5/6rev, 5'-AATATGTCGACTTACTTCTTGACCAAAGAAGCTTC-3'. All the amplified fragments were digested with SalI/BamHI and cloned into pGEX-4T-2 yielding in plasmids pGST-MBS1/2, pGST-MBS3/4, pGST-MBS3/4m3, pGST-MBS3/4m34, and pGST-MBS5/6. All the amplified sequences were verified by automated sequence analysis using the dideoxy chain termination kit (Amersham Biosciences) and the protocol provided by the manufacturer.
Expression and Purification of MBS FragmentsThe plasmids were transformed and expressed in Escherichia coli strain BL21(pREP4). Cells were grown at 37 °C in LB medium with 50 µg/ml ampicillin, harvested 2 h after induction with 0.1 mM isopropyl--D-thiogalactopyranoside, and lysed in 50 mM Tris-Cl (pH 8.0), 10 µg/ml lysozyme, and 2 mM dithiothreitol by sonicating the cells 20 times with an ultrasonic tip set to 50% power. After the addition of 1% Triton X-100, the lysate was centrifuged at 35,000 x g for 30 min, and the proteins were batch-purified using 50% GSH-Sepharose slurry (Amersham Biosciences) in phosphate-buffered saline + 5 mM dithiothreitol as described by the manufacturer. The purified MBSs were released from both GST and GSH-Sepharose in a single reaction step by 10 NIH units/ml of thrombin (Sigma) and subsequently analyzed by SDS-PAGE and silver staining (38). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad).
Surface Plasmon ResonanceSurface plasmon resonance experiments were performed using a Biacore Upgrade biosensor system (Biacore Inc., Uppsala, Sweden). Purified mutant or wt MBS3/4, MBS5/6, GST-MBS1/2, or GST were diluted to 50 µg/ml in 10 mM NaOAc, pH 4.6, and covalently coupled to the sensor surface by the standard N-hydroxysuccinimide/N-ethyl-N'-(dimethyl-aminopropyl-)carbodiimide activation chemistry according to the manufacturer's instructions. Fusion proteins were injected until 8003000 response units (RU) of wt and mutated MBS3/4, MBS5/6, GST-MBS1/2, and GST were coupled. These values corresponded to 0.84.8 ng/mm2 immobilized protein on the chip surface. Remaining free N-hydroxysuccinimide groups were saturated by a 6-min 1 M ethanolamine injection. The interaction analysis was performed at 37 °C in a flow buffer containing 2 mM ascorbate and 50 mM NaPi, pH 7.4, at a flow rate of 5 µl/min. The sequence of the Atox1-MBS interaction experiments was as depicted in Fig. 4: metal ions were first removed from the system by a 2-min wash with 1 mM EDTA, 0.1 mM bathocuproine disulfonate (BCS) in 50 mM NaPi, pH 7.4. The immobilized proteins on the chip were then reduced by 20 mM Tris-(2-carboxy-ethyl) phosphine (TCEP) in 10 mM sodium citrate, pH 3.0 for 4 min (39). When required, the chip was preloaded with copper using 0.01 mM CuCl2 in flow buffer. Subsequently, Atox1 was injected for 7 min. As described under "Results," the interaction event was monitored in real-time (see Fig. 4, inset), and the sensor chip was finally regenerated with 2 M NaCl. This washing step reset the RUs to the initial, baseline values.
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Surface Plasmon Resonance AnalysisTo estimate the apparent association and dissociation rate constants, we used linear transformation of the biosensor curves. The primary data were analyzed using the BIAevaluation 3.1 Software (Biacore Inc.), applying a Langmuir binding model (stoichiometry of 1:1) to calculate kon (association rate constant M1 s1), koff (dissociation rate constant, M1 s1), and KD (equilibrium constant) for the interaction of Atox1 with mutated or wt MBS fragments. For each concentration, kon and koff were obtained simultaneously from the equation KD = koff/kon. The goodness of the fit between the fitted curve and the experimental data was expressed as 2 according to the manufacturer's protocol. For good fitting to ideal data,
2 is of the same order of magnitude as the noise in RU, typically <10.
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RESULTS |
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To investigate kinetics of the interaction of Atox1 with the MBSs of the Menkes protein, we used real-time SPR analysis. This method has been widely used in several studies to analyze relatively stable receptor-ligand interactions (40), and in a recent study of transient, copper-dependent chaperone-acceptor interaction (30). SPR is based on a change in the refractive index of a light beam upon binding of a molecule in solution to a molecule immobilized on a dextran-coated chip surface. The data are expressed in RUs and are directly proportional to the mass of protein that is bound to the immobilized protein. A response of 1000 units corresponds to a change in surface protein concentration of 1 ng/mm2. In expectation of a transient and therefore low signal, we used GST-tagged MNK fragments purified over a GSH-Sepharose resin. The GST tag was removed by thrombin, and the purified fragments were analyzed on an SDS-polyacrylamide gel (Fig. 3). GST-MBS1/2 could be cleaved only in phosphate-buffered saline containing 0.005% SDS. Digestion of this slightly denatured protein with thrombin resulted in the release of two fragments of molecular mass of 26 kDa (data not shown). Because of near identical sizes and similar pI-values of GST and MBS1/2, we were unable to effectively isolate MBS1/2 and therefore used GST-MBS1/2 and GST as a control in the SPR experiments. All of the fragments had the expected molecular weights (Fig. 3). The 69-amino-acid, water-soluble chaperone Atox1 was chemically synthesized and analyzed as described under "Experimental Procedures."
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The purified MNK fragments were covalently coupled to the dextran surface of the sensor chip as outlined by the manufacturer. When Atox1 was immobilized on the chip, no interaction with any of the MBSs could be detected. This was also observed when the bacterial copper chaperone CopZ was coupled onto the chip (30) and was assigned to steric hindrance of the interaction caused by preferential covalent binding of the chaperone to the chip via its basic amino acids (see also "Discussion"). Between 3.0 and 4.8 ng of MNK fragments corresponding to 90 fmol/mm2 GST-MBS1/2, 160 fmol/mm2 wt, and mutant MBS3/4 and 200 fmol/mm2 MBS5/6 were immobilized on the dextran-coated surface. For the analysis of the interaction, we used a regeneration/binding/washing procedure as shown in Fig. 4. The presence of TCEP, which readily reduces oxidized thiol groups in proteins (39), and a flow buffer containing 2 mM ascorbate turned out to be crucial. When either TCEP or ascorbic acid was removed from the system, the RUs continuously increased over time in a linear fashion and, after an extensive regeneration wash, did not drop to the baseline, indicating non-specific accumulation of Atox1 on the chip surface (data not shown). When required, the MBSs were copper-loaded by a short (1 min) burst of 10 µM copper sulfate in flow buffer.
We first investigated interactions of the immobilized fragments GST, GST-MBS1/2, MBS3/4, and MBS5/6 with 10 µg/ml (1.35 µM) and 100 µg/ml (13.5 µM) Atox1 (Fig. 5, AD). There was no specific interaction detected when Atox1 was injected onto the GST surface (Fig. 5A, IIV). The slight increase in response units with 100 µg/ml Atox1 appeared to be non-specific because the relative response units dropped to zero when the injection cycle of the chaperone was finished. When 100 µg/ml Atox1 was injected onto a copper-loaded GST-MBS1/2-coated chip surface, we observed an increase of 100 RUs, indicating a binding to MBS1/2 (Fig. 5B, IV). This interaction was copper-dependent (Fig. 5B, compare I and III (copper) with II and IV (+copper)) and specific to Atox1 as 200 µg/ml (2.3 µM) of the amyloid precursor protein, a copper-binding protein suggested to be involved in copper homeostasis in the brain (41), only showed marginal interaction (Fig. 5B, gray curve). This subtle interaction may be due to the presence of a heavy metal binding site in amyloid precursor protein (42). Similar to MBS1/2, Atox1 interacted with MBS3/4 and MBS5/6 in a copper-dependent fashion (Fig. 5, C and D, compare I and III (copper) with II and IV (+copper)), and there was no interaction observed with 150 µg/ml (2.17 µM) bovine serum albumin (Fig. 5, C and D, curves in gray), a protein containing heavy metal binding sites (43), which was shown previously to refuse the acceptance of copper from the copper-loaded yeast homologue Atx1 (44).
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Mutations in the MBSs have been demonstrated to affect both copper binding (45) and Atox1-MBS interactions, as has been analyzed by yeast two-hybrid (Ref. 29 and this report). We therefore used purified MBS3/4 fragments with CXXC to SXXS mutations in MBS3 (MBS3/4m3) and Cys to Ser mutations in both MBS3 and MBS4 (MBS3/4m34) to investigate real-time interaction with Atox1 (Fig. 6). In line with previously published data (29) and yeast two-hybrid data from this report, we did not detect a significant interaction between Atox1 and the copper-loaded double-mutant MBS3/4m34 (Fig. 6B). There was some interaction observed between Atox1 and the single site mutant MBS3/4m3, indicating that Atox1 interacted with the single, copper-loaded MBS4 (Fig. 6A).
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Association (kon) and dissociation (koff) rate constants were determined by linear transformation of the sensorgrams according to the Langmuir binding model, which assumes that the MBS-Atox1 interaction is pseudo-first order. kon and koff were determined from linear regression analysis at varying concentrations of the ligand (10, 20, 40, 60, 100, and 150 µg/ml Atox1). When the MBSs were not preloaded with copper, we were not able to calculate association and dissociation rate constants. Therefore, kinetic parameters of copper-dependent interactions only, which were obtained by curve fitting using the BIAevaluation software version 3.0, are listed in Table I.
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Atox1 did not bind to a GST surface preloaded with copper (Fig. 5A), and therefore association and dissociation rates were not determined (indicated as NA (not analyzable) in Table I). Importantly, this indicated that GST alone did not contribute significantly to the binding of GST-MBS1/2 to Atox1. However, the association rate of Atox1 with GST-MBS1/2 was somewhat lower (0.12 x 103 M1 x sec1) than with MBS3/4 and MBS5/6 (1.64 x 103 M1 x sec1 and 0.89 x 103 M1 x sec1, respectively), which may be assigned to the bulky GST in the GST-MBS1/2 fragment. Dissociation occurred in a similar fashion for the two MNK fragments MBS3/4m3 and MBS5/6 (around 4 x 103 sec1) and was 2-fold lower for GST-MBS1/2 and MBS3/4 (around 2 x 103 sec1). These differences in the association and dissociation rate constants led to varying binding constants, indicating different binding affinities. However, due to the relatively weak and transient nature of the copper-dependent interactions, variations were observed in different experiments. Thus, the subtle differences in the equilibrium constants observed in these SPR experiments were considered to be non-significant.
CXXC to SXXS mutations in MBS3 did not significantly affect the association and dissociation rate constant of Atox1, indicating that both MBS3 and MBS4 may have similar affinities for the copper chaperone. CXXC to SXXS mutations in both MBS3 and MBS4 completely abolished the binding of Atox1 to the fragment MBS3/4m34 (Fig. 6B and in Table I, indicated as NA, not analyzable), which underlined the essentiality of the Cys residues for the chaperone-MBS interaction.
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DISCUSSION |
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Our results from both assays demonstrated a copper-dependent interaction of Atox1 with MBS14 of MNK. The -galactosidase activity in the yeast two-hybrid assay was minimal for MBS5/6, suggesting that Atox1 did not bind to these two MBSs. This result is in accordance with previously published two-hybrid data on the WND N terminus (29) and in apparent contradiction to the results obtained by SPR (Fig. 5D). Yeast two-hybrid analysis is based on an interaction-dependent reconstitution of a transcription factor in the yeast nucleus and thus is an indirect method for protein-protein interactions. High salt concentrations, varying pH, or instabilities caused by intermolecular interactions in the yeast nucleus may have a dramatic effect on the formation of a fully functional transcription factor and therefore may lead to false positive or false negative results. Data from yeast two-hybrid analysis should therefore be taken with caution and were here verified by a real-time protein-protein interaction method.
For over 10 years, SPR analysis has been used for the investigation of high affinity receptor-ligand interactions (40), and more recently, in several lower affinity binding studies (30, 47, 48). Relatively low affinity interactions (kon = 103 M1 s1) were observed when the MBSs were immobilized on the chip surface and the copper chaperone was injected, and not vice versa, when Atox1 was immobilized on the chip and the MBSs were injected. Clustered basic sites are exposed on the surface of copper chaperones, whereas the MBSs of the ATPases contain clusters of acidic residues (24, 49). These regions were predicted to be involved in direct electrostatic protein-protein interactions. Preferential immobilization of Atox1 to the chip surface by these basic regions may explain why an interaction was detected only when the MBSs but not the chaperone were covalently linked to the dextran layer of the chip surface. The validity of this "reversed approach" is underlined by an investigation on the complex formation and dissociation of the yeast homologue Atx1 with Ccc2, which was reversible, rapid, and with a half-life for the complex in the order of milliseconds (25). Another in vitro binding study also demonstrated that purified Cu(I)-Atx1 donated its metal ion cargo to the N-terminal MBS in a direct and reversible manner (44).
The kinetic data calculated from our experiments proposed similar affinities for all the MBSs with Atox1 (Table I) with binding constants in the range of 14 x 106 M for MBS36 and 19 x 106 M for MBS1/2. However, the apparent lower affinity of MBS1/2 with Atox1 may be attributed to sterical hindrance of the interaction due to the bulky GST attached at the N terminus.
The hyperbolic graphs suggested a negative Atox1 cooperativity with all the MBSs and therefore a functional identity in terms of copper-dependent binding of the chaperone. The negative cooperativity is strengthened by the association and dissociation rate constants obtained with MBS3/4m3: the affinity of this single site mutant was not significantly altered as compared with the wild type fragment.
The non-cooperative interaction of the chaperone with all the MBSs may contradict the model of copper acceptance and delivery, as suggested by Huffman et al. (46). In this report, the structures of the MBSs were generated by homology modeling utilizing the coordinates of silver-loaded MBS4 (20) as a template. As the MBS5 and MBS6 of the human copper ATPase are linked by only 2 or 3 amino acids, Huffman et al. (46) proposed that domains 5 and 6 may act in concert and as copper acceptors that receive the cargo from other cytosolic domains of the P-type ATPases. However, our results were generated with pairs of MBSs and did not include the full-length N terminus. We therefore cannot entirely rule out the possibility that Atox1 transfers copper to the N-terminal MBSs and from there is then directly passed onto MBS5 and MBS6. To elucidate this model, SPR experiments involving the full-length N terminus and mutants thereof are under investigation.
In conclusion, we here showed by real-time surface plasmon resonance that all the metal binding sites of the Menkes protein were involved in binding of copper-loaded Atox1. The interaction of Atox1 with the MBSs was transient, of non-cooperative nature, and indicated similar binding constants for all the MBSs in the order of 105-106 M. Therefore, a functional difference of the MBSs in the MNK N terminus may not be induced by varying affinities of the metallochaperone Atox1 to the MBSs of MNK.
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FOOTNOTES |
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¶ A recipient of a fellowship from the Roche Research Foundation.
** Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 61-3-9251-7329; Fax: 61-3-9251-7328; E-mail: jmercer{at}deakin.edu.au.
1 The abbreviations used are: MNK, Menkes protein; WND, Wilson protein; MBS, metal binding site; SPR, surface plasmon resonance; wt, wild type; GST, glutathione S-transferase; RU, response units; BCS, bathocuproine disulfonate; TCEP, Tris-(2-carboxy-ethyl)phosphine.
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ACKNOWLEDGMENTS |
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REFERENCES |
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