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• W2085777790 abstract "Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys1010–Lys1325 segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys1010–Lys1325fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed. Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys1010–Lys1325 segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys1010–Lys1325fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed. Wilson's disease Wilson's disease protein Menkes disease protein N-terminal domain of the Wilson's disease protein ATP-binding domain of the Wilson's disease protein ATP-binding domain of Na+,K+-ATPase maltose-binding protein maltose-binding protein fusion of N-WNDP 2′,3′-O-(2,4,6,-trinitrophenyl)-ATP antibody isopropyl-β-d-thiogalactopyranoside nickel-nitrilotriacetic acid 4-morpholinepropanesulfonic acid guanidine hydrochloride Copper is an essential trace element that serves as a cofactor for a variety of key metabolic enzymes, such as tyrosinase, cytochromec oxidase, superoxide dismutase and many others. Inborn copper deficiency, known as Menkes disease, leads to a dramatic decrease in the activity of these enzymes, causing severe developmental delays, poor temperature control, and defects of connective and vascular tissues. Recent genetic studies linked this disease to various mutations in the ATP7A gene located on the X chromosome (1Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1217) Google Scholar, 2Verga V. Hall B.K. Wang S.R. Johnson S. Higgins J.V. Glover T.W. Am. J. Hum. Genet. 1991; 48: 1133-1138PubMed Google Scholar). The ATP7A gene product, or Menkes disease protein, is a copper-transporting P-type ATPase, the primary function of which is to export dietary copper from intestinal cells for further delivery to various tissues. Copper overload is as deleterious to cells as copper deficiency, presumably because of the ability of copper to participate in reactions that generate highly reactive oxygen species (3Stohs S.J. Bagchi D. Free Radic. Biol. Med. 1995; 18: 321-336Crossref PubMed Scopus (3547) Google Scholar). Wilson's disease (WD)1 is an autosomal disorder associated with vast accumulation of copper in the liver, brain, and kidneys (4Scheinberg I.H. Sternlieb I. Smith Jr., L.H. Wilson's Disease: Major Problems in Internal Medicine. W. B. Saunders, Philadelphia, PA1984Google Scholar). The disease is caused by mutations in the ATP7B gene that encodes another copper-transporting P-type ATPase (5Petrukhin K. Lutsenko S. Chernov I. Ross B.M. Kaplan J.H. Gilliam T.C. Hum. Mol. Genet. 1994; 3: 1647-1656Crossref PubMed Scopus (306) Google Scholar, 6Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1703) Google Scholar, 7Yamaguchi Y. Heiny M.E. Gitlin J.D. Biochem. Cell Biol. Commun. 1993; 197: 271-277Google Scholar). The Wilson's disease protein (WNDP) and Menkes disease protein (MNKP) share over 50% sequence similarity and are likely to have similar mechanistic properties (8Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). All P-type ATPases can be divided into five major branches based on their substrate specificity (9Axelsen L.B. Palmgren M.G. J. Mol. Evolution. 1998; 46: 84-101Crossref PubMed Scopus (752) Google Scholar). WNDP and MNKP belong to the P1-(CPx) subfamily of the P-type ATPases (10Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (417) Google Scholar, 11Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (417) Google Scholar). All members of this subfamily utilize energy of ATP hydrolysis to transport either transition or heavy metals across cell membranes, and all have characteristic metal-binding motifs at the N-terminal portion of the molecule (Fig. 1). In contrast, the P2-ATPases are involved in transport of alkali or alkali earth metals and, as a rule, lack the characteristic metal-binding sites at their N termini. Na+,K+-ATPase is a typical representative of the P2-ATPase subfamily with known nucleotide binding properties, and it has been utilized in this study for comparative purposes. Although mechanistic properties of the P2-ATPases are well studied, very few members of the P1-ATPase subfamily have been functionally characterized (12Rensing C. Fan B. Sharma R. Mitra B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 652-656Crossref PubMed Scopus (402) Google Scholar, 13Sharma R. Rensing C. Rosen B.P. Mitra B. J. Biol. Chem. 2000; 275: 3873-3878Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 14Wyler-Duda P. Solioz M. FEBS Lett. 1996; 399: 143-146Crossref PubMed Scopus (19) Google Scholar, 15Tsai K.J. Linet A.L. Arch. Biochem. Biophys. 1993; 305: 267-270Crossref PubMed Scopus (41) Google Scholar). ATP-dependent transport has been recently demonstrated for MNKP (16Voskoboinik I. Brooks H. Smith S. Shen P. Camakaris J. FEBS Lett. 1998; 435: 178-182Crossref PubMed Scopus (62) Google Scholar), but the detailed molecular mechanism for either MNKP or WNDP remains unknown. Location of the copper-binding sites in the N-terminal domain, separately from the ATP-binding region and the transmembrane cation-translocation pathway (Fig. 1), suggests that the ATP-driven copper transport by WNDP and MNKP is likely to involve cooperation between several protein domains. The results obtained in this work substantiate this hypothesis. At present, purification and reconstitution procedures for either MNKP or WNDP are unavailable, severely limiting the detailed analysis of their structure and function. However, the use of recombinant proteins corresponding to the putative functional domains of WNDP and MNKP proved to be very informative. Recently, we and others produced and biochemically characterized the recombinant N-terminal copper-binding domain of both WNDP and MNKP (17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 18DiDonato M. Narindrasorasak S. Forbes J.R. Cox D.W. Sarkar B. J. Biol. Chem. 1997; 272: 33279-33282Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19Harrison M.D. Meier S. Dameron C.T. Biochim. Biophys. Acta. 1999; 1453: 254-260Crossref PubMed Scopus (21) Google Scholar, 20DiDonato M. Hsu H.F. Narindrasorasak S. Que Jr., L. Sarkar B. Biochemistry. 2000; 39: 1890-1896Crossref PubMed Scopus (105) Google Scholar). The studies revealed that this 70-kDa domain binds copper specifically in vivo and in vitro with stoichiometry of 5.5–6 copper/domain, suggesting that each of the six GMTCXXC motifs present in this domain participate in copper coordination (17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 18DiDonato M. Narindrasorasak S. Forbes J.R. Cox D.W. Sarkar B. J. Biol. Chem. 1997; 272: 33279-33282Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Furthermore, it has been shown that copper binds to Cys residues in the GMTCXXC motif as Cu+1 via novel linear coordination (20DiDonato M. Hsu H.F. Narindrasorasak S. Que Jr., L. Sarkar B. Biochemistry. 2000; 39: 1890-1896Crossref PubMed Scopus (105) Google Scholar, 21Ralle M. Cooper M.J. Lutsenko S. Blackburn N.J. J. Am. Chem. Soc. 1998; 120: 13525-13526Crossref Scopus (59) Google Scholar). Present studies demonstrate that in addition to its copper binding function the N-terminal domain of WNDP may play an important regulatory role. In the case of WNDP and MNKP, analysis of recombinant domains offers another important advantage: it allows us to assess individually their overall folding and functional characteristics. This information is particularly important if one wants to understand how mutations in certain regions of WNDP and MNKP alter their specific properties. A number of the WD-causing mutations have been identified in the Lys1010–Lys1325 region of WNDP (Fig. 1) (22Shah A.B. Chernov I. Zhang H.T. Ross B.M. Das K. Lutsenko S. Parano E. Pavone L. Evgrafov O. Ivanova-Smolenskaya I.A. Anneren G. Westermark K. Urrutia F.H. Penchaszadeh G.K. Sternlieb I. Scheinberg I.H. Gilliam T.C. Petrukhin K. Am. J. Hum. Genet. 1997; 61: 317-328Abstract Full Text PDF PubMed Scopus (307) Google Scholar,23Thomas G.R. Forbes J.R. Roberts E.A. Walshe J.M. Cox D.W. Nat. Genet. 1995; 9: 210-217Crossref PubMed Scopus (491) Google Scholar). For only one of them, His1070 → Gln, there is evidence suggesting that the substitution of His for Gln is associated with the WNDP misfolding in a cell (24Payne A.S. Kelly E.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10854-10859Crossref PubMed Scopus (188) Google Scholar). The specific molecular consequences for most of the other mutations are still unknown. Consequently, the ability to analyze folding and functional properties of normal and mutant domains of WNDP would be very valuable. In this paper we describe several biochemical procedures that can be utilized for these purposes. The WNDP segment including the amino acid residues Lys1010–Lys1325 and the homologous region of MNKP were predicted to act as the ATP-binding domains (ATP-BDs) of these proteins. The suggestion was based on the presence in this region of several highly conserved motifs (Fig. 1) previously shown to be involved in ATP binding and hydrolysis in such P2-type ATPases as Na+,K+-ATPase and Ca2+-ATPase. At the same time, the overall homology between the corresponding segments of WNDP (P1-ATPase) and Na+,K+-ATPase or Ca2+-ATPase (P2-ATPases) is only 18–23%, suggesting that the specific nucleotide binding properties and the regulatory mechanisms modulating binding of ATP may differ for the P1- and P2-ATPases. Our analysis of the ATP-binding domain of WNDP supports this hypothesis. In this paper, we provide experimental evidence that the recombinant Lys1010–Lys1325 fragment of WNDP forms an independently folded ATP-BD, which has distinct nucleotide binding properties and measurable ATPase activity, that ATP-BD interacts with the N-terminal copper-binding domain (N-WNDP) in a copper-dependent manner, and that the copper-dependent domain-domain interactions induce conformational changes in ATP-BD. These results shed a light on some steps in molecular mechanism of WNDP and highly homologous MNKP. The aN-WND is a polyclonal antibody (Ab) developed against the peptide GMTCASCVHNIE, which corresponds to the sixth metal-binding repeat Gly572–Glu583 in the N-WNDP. The polyclonal Ab a-ABD was raised against the purified recombinant ATP-BD (for cloning and expression of ATP-BD, please see below). The Ab against the His tag was purchased from Qiagen, anti-maltose-binding protein antibodies were obtained from NeoMarkers. The construct encoding the ATP-binding loop of rat Na+,K+-ATPase α1 subunit (residues Lys354–Lys774) was kindly provided to us by Dr. C. Gatto (Oregon Health Sciences University). We will further refer to protein produced using this construct as the M4M5 loop. The plasmid encoding the His-tagged C-terminal domain of the small conductance calcium-activated potassium channel (ABC domain) was a gift from J. P. Adelman's laboratory (Vollum Institute). The putative ATP-BD was expressed as a fusion protein with a His tag at the N-terminal portion of the molecule. To generate the expression construct, the segment of the full-length WNDP cDNA encoding the amino acid residues Lys1010–Lys1325 was amplified by polymerase chain reaction using the following primers WD-ATP-fwd, 5′-GAATTCCATATGAAGGGAGGCAAG-3′, and WD-ATP-Rew, 5′-GAATTCCATATGCTAGCGTATCCTTCGGAC-3′. The procedure also created theNdeI sites that were used to clone the polymerase chain reaction product into the PET-28b vector (Novagen). (The lack of unwanted mutations in the obtained construct was verified by automatic DNA sequencing.) The construct was used to transform Escherichia coli BL21 (DE3) cells; the protein expression was induced by treatment of cells with either 0.1 mm IPTG (for purification from soluble cell fraction) or 1 mm IPTG (for purification from inclusion bodies) for 1 h at room temperature. E. coli cells were resuspended in lysis buffer prepared by dissolving 1 Complete protease inhibitor tablet (Roche Molecular Biochemicals) in 50 ml of 50 mm Tris, 500 mmNaCl, pH 7.5, and then lysed by passing twice through the French press (American Instrument Co.) at 20,000 p.s.i. To decrease viscosity, lysate was additionally passed two times by syringe through a medium gauge needle. Soluble proteins were separated from the insoluble fraction following centrifugation of the lysate at 30,000 ×g for 20 min. To analyze the distribution of ATP-BD between soluble and insoluble fractions, insoluble protein was resuspended in the lysis buffer to the same volume as soluble protein. Identical aliquots were taken from both fractions and separated by 10% Laemmli gel, and then proteins were transferred to Immobilon-P membrane as described (25Noll M. Petrukhin K. Lutsenko S. J. Biol. Chem. 1998; 273: 21393-21401Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The amount of ATP-BD in each fraction was determined by staining of the blot with the a-ABD Ab (dilution 1:20,000) followed by densitometry of immunostained bands. The solubility of ATP-BD depended on the level of expression. Following induction with 1 mmIPTG, almost all ATP-BD was quickly deposited into the inclusion bodies. When protein expression was significantly lowered by changing the IPTG concentration from 1.0 to 0.1 mm, the overall amount of produced ATP-BD decreased greatly, but proportionally more protein was found in the soluble fraction. Analysis of the distribution of ATP-BD revealed that at 0.1 mm IPTG ∼20% of the produced ATP-BD was soluble, and only this protein was used for characterization of its functional properties. The expression of the M4M5 loop and the ABC domain and preparation of soluble fractions containing these proteins were carried out as described below for ATP-BD. Purification of the His6-tagged proteins (ATP-BD, M4M5 loop, and ABC domain) by Ni2+ affinity chromatography was carried out at room temperature except where indicated. The soluble fraction of lysate was loaded on Ni-NTA-agarose resin (Qiagen); the amount of Ni-NTA-agarose to be used was determined based on the expected yield of purified protein (200 μl slurry/mg for M4M5 loop, 1 ml/mg for ATP-BD). Imidazole was added to the lysate to 50 mm prior to binding. Following a 1-h incubation of the lysate with constant agitation, the resin was applied on a column and washed with at least 30 volumes of wash buffer (50 mmimidazole, 50 mm Tris, 500 mm NaCl, pH 7.5). The target proteins were then eluted in several fractions of elution buffer (200 mm imidazole, 50 mm Tris, 500 mm NaCl, pH 7.5). Protein concentration was determined by the method of Bradford (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). 0.5–2.0 μg of protein were loaded onto a 10% SDS-polyacrylamide gel, and protein purity was ascertained by Coomassie staining. For analysis of folding and nucleotide binding properties, the protein was dialyzed twice against 50 mmMOPS pH 7.5 buffer containing either 150 or 500 mmNaCl. The recombinant N-terminal domain of WND tagged to the maltose-binding protein (N-WND-MBP) in its copper-bound and copper-free form was overexpressed and purified as described previously (17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The recombinant 42-kDa maltose-binding protein (MBP) was generated using the pMal-c vector (New England BioLabs) and purified using the same protocol (17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The ATP-BD folding was analyzed by using the intrinsic tryptophan fluorescence. The measurements were performed on a model PTI-QM1 (Photon Technology International) fluorimeter. Emission spectra were recorded from 305 to 405 nm (bandwidth, 2 nm) with an excitation wavelength of 295 nm (bandwidth, 3 nm) for 1.25 μm ATP-BD solution in 50 mmMOPS, pH 7.5, 500 mm NaCl in the absence or presence of 6m guanidine hydrochloride (GuHCl). The solution ofl-tryptophan in the above buffer at concentration similar to concentration of ATP-BD was used in these experiments as a standard. For experiments in which the effect of nucleotides on the Trp fluorescence was tested, the excitation was set at 295 nm, emission was set at 345 nm, and 800 μl of the same protein solution was titrated with 2-μl aliquots of ATP or AMP to obtain final concentration ranging from 0 to 800 μm. Identical aliquots of buffer were added to control sample to account for a change in the fluorescence because of dilution; the final increase in volume was less than 2%. Fluorescence changes upon binding of TNP-ATP to ATP-BD or the M4M5 loop were measured at room temperature in transparent polystyrene 96-well plates (Falcon, Becton Dickinson) using a FLUOstar microplate reader (BMG Labtechnologies Inc.). The excitation was set at 390 nm, and the emission was recorded at 538 nm. The reaction was carried out in 50 mm MOPS pH 7.5 buffer containing various NaCl concentration (20–500 mm). The 1-μl aliquots of the TNP-ATP stock solution were added to 100 μl of a 5 μm protein solution for final concentrations ranging from 0 to 40 μm. (The increase in the reaction volume did not exceed 10% and was taken into account.) To determine the background fluorescence, identical TNP-ATP titrations were made in 50 mm MOPS pH 7.5 buffer containing 20–500 mm NaCl. The apparent binding affinities of ATP-BD and M4M5 loop for TNP-ATP were determined after subtracting the background TNP-ATP fluorescence using SigmaPlot software package. For most experiments, NaCl concentration was kept at 20 mm, because both the TNP-ATP affinity and the fluorescence increase upon TNP-ATP binding to ATP-BD were higher at lower salt concentrations. (For example, the K a for TNP-ATP at 20 mmNaCl was 1.89 ± 0.72 μm, whereas at 500 mm the K a was 6.3 μm ± 1.5.) These differences in the TNP-ATP binding parameters are likely to reflect small changes in the nucleotide-binding pocket, because changes in NaCl concentrations do not have noticeable effect on overall protein folding as evidenced by the Trp fluorescence spectra. 2R. Tsivkovskii, B. C. MacArthur, and S. Lutsenko, unpublished data. The effect of N-WNDP on TNP-ATP binding was analyzed as described above using equimolar amounts of ATP-BD and either copper-free or copper-bound N-WNDP in 50 mm MOPS, 20 mm NaCl, pH 7.5 buffer. The background TNP-ATP binding to N-WNDP was similar for copper-free and copper-bound proteins and was subtracted. The displacement of TNP-ATP by nucleotides was analyzed using PTI-QM1 fluorimeter with excitation set at 410 nm and emission set at 545 nm. For these experiments, TNP-ATP at 0.5 μm was mixed with 2.5 μm protein solution in 50 mm MOPS pH 7.5 buffer, containing 500 or 20 mm NaCl and then titrated with ATP, with AMP (0–800 μm) or with the same volumes of buffer as a control. The protein fluorescence before TNP-ATP addition served as a background and was subtracted. The ATPase Activity of ATP-BD was measured using the EnzChek™ Phosphate Assay (Molecular Probes). In this assay, inorganic phosphate released during ATP-hydrolysis is utilized by purine nucleoside phosphorylase to convert the substrate 2-amino-6-mercapto-7 methylpurine riboside to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. This leads to a shift in maximum absorbance from 330 nm for the substrate to 360 nm for the product that can be monitored spectrophotometrically. For the ATPase measurements, the purified ATP-BD was dialyzed against the reaction buffer (20 mm Tris-HCl, 1 mmMgCl2, 200 mm NaCl, pH 7.5) and then incubated in 10–20 μl of volume with 1 mm ATP at protein concentration of 0.1 mg/ml for 15 min at 37 °C. The reaction was stopped by transferring samples on ice, and the amount of released phosphate was determined using the Molecular Probes protocol. The control His tag protein corresponding to the fragment of the WNDP copper-binding domain was expressed in the same strain of cells as ATP-BD and then purified under identical conditions. The elution fractions containing this protein or just buffer were used as negative controls. The domain-domain interactions were evaluated by an optimized quantitative copurification procedure. In preparation for the interaction experiment, a preliminary purification of ATP-BD and the control His tag proteins was carried out to determine the relative molar concentrations of the target proteins in their respective lysates. The amount of each His-tagged protein was quantified by densitometry of the Coomassie-stained gel using bovine serum albumin as a standard. The amount of ATP-BD in the soluble portion of the lysate was lower than that of the M4M5 loop and the ABC protein, consequently the soluble lysate from the nontransformed BL21 cells was used to dilute the M4M5 loop and ABC lysates appropriately and bring the concentration of all His tag proteins to the same molar concentration. Miniature affinity chromatography columns were constructed using glass wool, standard 1-ml pipette tips, and 5 μl of Qiagen Ni-NTA agarose resin. The equal amounts of the primary His tag proteins (ATP-BD, M4M5 loop, and ABC) or lysate from nontransformed BL21 were incubated with the Ni-NTA resin. The resin was washed as described in the purification procedure above, and then the secondary proteins (NWND-MBP − Cu, NWND-MBP + Cu, or MBP) were run over the columns. After two additional wash steps with 200 bed volumes of buffer, the bound proteins were eluted in 200 mm imidazole. The presence of proteins in the elution fractions was detected by Western blotting using the appropriate Ab. The anti-MBP Ab was used at a 1:40,000 to 1:100,000 dilution, the a-ABD and a-N-WND were used at 1:20,000 dilution, and protein bands were visualized using Pierce SuperSignal chemiluminescence procedure. For domain-domain interaction assay on amylose resin, cells expressing N-WNDP with or without copper (for expression conditions see Ref. 17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) from a 1-liter culture were lysed in 40 ml of PB buffer (50 mm sodium phosphate, pH 7.5, 500 mm NaCl) + 1 tablet of complete protease inhibitor using French press. After clearing lysate by centrifugation aliquots were purified as described (17Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) to determine the amount of lysates needed to obtain identical quantities of copper-free and copper-bound N-WNDP. The level of expression, solubility, and the yield of copper-free and copper-bound N-WNDP were very similar; therefore, similar amount of cell lysates have been used for the interaction experiments. Usually, 50 μl of amylose resin slurry (1:1) was added to 150 μl of lysate diluted till 500 μl with PB buffer, and the mixture was incubated on ice for 20 min with occasional stirring. Then resin was washed three times with 500 μl of PB buffer and once with 500 μl of TB buffer (50 mm Tris-HCl, pH 7.5, 500 mm NaCl). Next, 1 μg of ATP-BD in 100 μl of TB buffer was added to the resin and incubated at room temperature for 15 min. The unbound ATP-BD was removed, and the resin was washed twice with 500 μl of TB buffer. Finally, all proteins were eluted from the resin with 60 μl of PB buffer containing 10 mm maltose. The elution fractions were analyzed for the presence of N-WND or ATP-BD by immunoblotting using appropriate antibodies. To test the effect of N-WND on the nucleotide binding properties of ATP-BD, TNP-ATP at 0.5 μm was mixed with 2.5 μm ATP-BD solution in 50 mm MOPS, 20 mm NaCl, pH 7.5 buffer and then 2.5 μm of either copper-free or copper-bound N-WND-MBP was added to the mixture. The mixture was then titrated with ATP, AMP (0–800 μm) or with the same volumes of buffer as a control using PTI-QM1 fluorimeter with excitation set at 410 nm and emission set at 545 nm. The protein fluorescence before TNP-ATP addition served as a background and was subtracted. The recombinant Lys1010–Lys1325 fragment, corresponding to the putative ATP-BD of WNDP, was produced in E. coli as a His tag fusion protein following induction of protein expression with IPTG (Fig. 2 A). The calculated molecular mass of the ATP-BD His tag fusion, 38 kDa, agreed well with the molecular weight of the expressed protein (Fig. 2). Soluble ATP-BD was purified to about 95% of homogeneity (Fig. 2 B), and the yield of purified protein (200–500 μg from 1 liter of the cell culture), although limited, was sufficient to determine the major functional characteristics of this putative ATP-binding fragment. ATP-BD has a single Trp residue at the position corresponding to Trp1154 of the full-length WNDP (Fig. 1). In general, Trp fluorescence is very sensitive to local environment, a property that can be utilized to monitor protein folding or protein interactions with ligands. To determine whether the purified ATP-BD is folded, the local environment around the Trp1154 residue was examined by comparing the steady-state fluorescence emission spectrum of ATP-BD with the spectrum of free l-tryptophan present at the same molar concentration. As shown in Fig.3, the maximum emission wavelength (λmax) for ATP-BD is 330 nm in contrast to λmax for free l-Trp (348 nm), indicating that the purified ATP-BD is folded and that the tryptophan residue in folded ATP-BD is buried. In agreement with this conclusion, unfolding of ATP-BD with 6 m GuHCl is accompanied by a shift of λmax to 348 nm, which is identical to the maximum of the solvent-exposed tryptophan (Fig. 3). Overall, ATP-BD is relatively resistant to treatment with denaturing reagents: at least 2.0m GuHCl was required to observe some red shift in the λmax, and in the presence of 6 m GuHCl the maximum was fully shifted to 348 nm. Additions of ATP or AMP up to 800 μm produced no modifications in the intensity or position of the λmax for Trp fluorescence, suggesting a lack of changes in the Trp environment (data not shown). To determine whether the recombinant Lys1010–Lys1325 fragment binds nucleotides, an ATP analogue, TNP-ATP, was utilized. TNP-ATP is often employed for characterization of the ATP-binding proteins because of its unique fluorescent properties. TNP-ATP in solution is weakly fluorescent, but upon binding to proteins its fluorescence enhances substantially (27Hiratsuka T. Biochim. Biophys. Acta. 1982; 719: 509-517Crossref PubMed Scopus (51) Google Scholar, 28Nakamoto R.K. Inesi G. J. Biol. Chem. 1984; 259: 2961-2970Abstract Full Text PDF PubMed Google Scholar, 29Gatto C. Wang A.X. Kaplan J.H. J. Biol. Chem. 1998; 273: 10578-10585Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 30Capieaux E. Rapin C. Thines D. Dupont Y. Goffeau A. J. Biol. Chem. 1993; 268: 21895-21900Abstract Full Text PDF PubMed Google Sch" @default.
• W2085777790 created "2016-06-24" @default.
• W2085777790 creator A5038195252 @default.
• W2085777790 creator A5074602236 @default.
• W2085777790 creator A5079179187 @default.
• W2085777790 date "2001-01-01" @default.
• W2085777790 modified "2023-10-16" @default.
• W2085777790 title "The Lys1010–Lys1325 Fragment of the Wilson's Disease Protein Binds Nucleotides and Interacts with the N-terminal Domain of This Protein in a Copper-dependent Manner" @default.
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