The Distinct Functional Properties of the Nucleotide-binding Domain of ATP7B, the Human Copper-transporting ATPase
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m404553200
ISSN1083-351X
AutoresClinton T. Morgan, Ruslan Tsivkovskii, Yuri Kosinsky, Roman G. Efremov, Svetlana Lutsenko,
Tópico(s)Ion Transport and Channel Regulation
ResumoCopper transport by the P1-ATPase ATP7B, or Wilson disease protein (WNDP), 1The abbreviations used are: WNDP, Wilson disease protein; WD, Wilson disease; P-domain, phosphorylation domain; N-domain, nucleotide-binding domain; MNKP, Menkes disease protein; wt, wild type; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)ethanesulfonic acid; ZntA, zinc-transporting ATPase.1The abbreviations used are: WNDP, Wilson disease protein; WD, Wilson disease; P-domain, phosphorylation domain; N-domain, nucleotide-binding domain; MNKP, Menkes disease protein; wt, wild type; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)ethanesulfonic acid; ZntA, zinc-transporting ATPase. is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P2-ATPases are not conserved within the P1-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg2+-independent manner with a relatively high affinity of 75 μm, compared with millimolar affinities observed for the P2-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P1-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P1-ATPases are distinct from those of the P2-ATPases. Copper transport by the P1-ATPase ATP7B, or Wilson disease protein (WNDP), 1The abbreviations used are: WNDP, Wilson disease protein; WD, Wilson disease; P-domain, phosphorylation domain; N-domain, nucleotide-binding domain; MNKP, Menkes disease protein; wt, wild type; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)ethanesulfonic acid; ZntA, zinc-transporting ATPase.1The abbreviations used are: WNDP, Wilson disease protein; WD, Wilson disease; P-domain, phosphorylation domain; N-domain, nucleotide-binding domain; MNKP, Menkes disease protein; wt, wild type; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)ethanesulfonic acid; ZntA, zinc-transporting ATPase. is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P2-ATPases are not conserved within the P1-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg2+-independent manner with a relatively high affinity of 75 μm, compared with millimolar affinities observed for the P2-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P1-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P1-ATPases are distinct from those of the P2-ATPases. The Wilson disease protein (WNDP) is a key regulator of copper homeostasis in a number of tissues, particularly the liver, brain, and kidneys (1Lutsenko S. Efremov R.G. Tsivkovskii R. Walker J.M. J. Bioenerg. Biomembr. 2002; 34: 351-362Crossref PubMed Scopus (60) Google Scholar, 2Fatemi N. Sarkar B. J. Bioenerg. Biomembr. 2002; 34: 339-349Crossref PubMed Scopus (37) Google Scholar). WNDP transports copper from cytosol across cell membranes, using the energy of ATP hydrolysis. Under basal conditions, WNDP delivers copper to enzymes within the secretory pathway; it is also essential for cellular copper excretion when copper concentrations are elevated (1Lutsenko S. Efremov R.G. Tsivkovskii R. Walker J.M. J. Bioenerg. Biomembr. 2002; 34: 351-362Crossref PubMed Scopus (60) Google Scholar, 2Fatemi N. Sarkar B. J. Bioenerg. Biomembr. 2002; 34: 339-349Crossref PubMed Scopus (37) Google Scholar, 3Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (176) Google Scholar). Mutations in WNDP result in marked accumulation of copper in the cytosol and a severe hepatoneurological disorder known as Wilson disease (WD) (4Gitlin J.D. Gastroenterology. 2003; 125: 1868-1877Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 5Loudianos G. Gitlin J.D. Semin. Liver Dis. 2000; 20: 353-364Crossref PubMed Scopus (146) Google Scholar). The clinical manifestations of WD are diverse (6Riordan S.M. Williams R. J. Hepatol. 2001; 34: 165-171Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar); however, the specific contributions of various WD mutations to phenotypic diversity remain poorly understood (7Cox D.W. Moore S.D. J. Bioenerg. Biomembr. 2002; 34: 333-338Crossref PubMed Scopus (103) Google Scholar). Elucidating the consequences of mutations on WNDP structure and function is the first step toward a better understanding of molecular mechanisms underlying WD. In addition, an intriguing connection has recently been made between overexpression of WNDP and increased resistance of cells to the anticancer drug cisplatin (8Katano K. Safaei R. Samimi G. Holzer A. Rochdi M. Howell S.B. Mol. Pharmacol. 2003; 64: 466-473Crossref PubMed Scopus (106) Google Scholar, 9Nakayama K. Kanzaki A. Ogawa K. Miyazaki K. Neamati N. Takebayashi Y. Int. J. Cancer. 2002; 101: 488-495Crossref PubMed Scopus (128) Google Scholar, 10Komatsu M. Sumizawa T. Mutoh M. Chen Z.S. Terada K. Furukawa T. Yang X.L. Gao H. Miura N. Sugiyama T. Akiyama S. Cancer Res. 2000; 60: 1312-1316PubMed Google Scholar). These findings point to a role for WNDP as a potential pharmacological target and further emphasize the need for a better understanding of the protein's structure, function, and regulation. At present, such structural and biochemical information on WNDP is very limited. WNDP belongs to the large family of the P-type ATPases and displays the key catalytic properties expected for the members of this family. In particular, WNDP hydrolyzes ATP to form a transient phosphorylated intermediate at the invariant aspartate located in the DKTG sequence, a signature motif of the P-type ATPase (Fig. 1). Copper, the transported ion, markedly stimulates the reaction (11Tsivkovskii R. Eisses J.F. Kaplan J.H. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The first crystal structure of a P-type ATPase, Ca2+-ATPase of sarcoplasmic reticulum, was recently solved (12Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar, 13Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar). The structure yielded important information about the organization and conformational flexibility of this class of transporters. Ca2+-ATPase was shown to be composed of several functional domains: the ATP-binding domain; the transmembrane domain, containing sites for transported ions; and the actuator domain, which is critical for phosphatase activity and conformational transitions during the catalytic cycle (14Toyoshima C. Nomura H. Sugita Y. FEBS Lett. 2003; 555: 106-110Crossref PubMed Scopus (58) Google Scholar, 15Clausen J.D. Vilsen B. McIntosh D.B. Einholm A.P. Andersen J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2776-2781Crossref PubMed Scopus (62) Google Scholar). The ATP-binding domain of the P-type ATPases was shown to consist of two domains: the phosphorylation domain (the P-domain) containing the DKTG sequence and the nucleotide-binding domain (the N-domain), which contributes to ATP coordination. The P-type ATPases are divided into five subfamilies based on their ion specificity and structural characteristics (16Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (757) Google Scholar). WNDP is a member of the P1B-ATPase subfamily (16Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (757) Google Scholar, 17Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). The P1-ATPases share very limited sequence homology with Ca2+-ATPase or other P2-ATPases. In fact, residues strictly conserved among all P-type ATPases are almost exclusively limited to the P-domain and the actuator domain (Fig. 1A). The P-domain and actuator domain play essential roles in catalysis; thus, it is not surprising that these two domains contain the sequence motifs found in all P-type ATPases. The N-domain is equally important, because it is involved in nucleotide binding. Therefore, it is intriguing that the amino acid residues known to participate in ATP coordination in the N-domain of Ca2+-ATPase and other P2-ATPases are not conserved in the structure of the P1-ATPases. This observation suggests that the coordination environment of the nucleotide in these two P-type ATPase subfamilies could be quite different. Although there is little sequence similarity between the N-domains of the P1- and P2-ATPases, the sequence of the N-domain is well conserved within the P1-ATPase subgroup. A number of the residues are highly conserved or invariant (Fig. 1B), suggesting their potentially important roles in the structure or function of the P1-ATPase N-domains. In the WNDP sequence, these residues include Glu-1064, Ser-1067, His-1069, Pro-1070, Gly-1101, and Gly-1103. It is noteworthy that invariant His-1069 is the site of the most frequent disease-causing mutation, an observation that further emphasizes the functional significance of this residue. Previous studies characterized the effect of mutations of His-1069, or equivalent histidines, on the functional activity of WNDP and several homologous P1-ATPases (18Bissig K.D. Wunderli-Ye H. Duda P.W. Solioz M. Biochem. J. 2001; 357: 217-223Crossref PubMed Scopus (50) Google Scholar, 19Okkeri J. Bencomo E. Pietila M. Haltia T. Eur. J. Biochem. 2002; 269: 1579-1586Crossref PubMed Scopus (16) Google Scholar, 20Okkeri J. Laakkonen L. Haltia T. Biochem. J. 2004; 377: 95-105Crossref PubMed Google Scholar, 21Voskoboinik I. Mar J. Camakaris J. Biochem. Biophys. Res. Commun. 2003; 301: 488-494Crossref PubMed Scopus (19) Google Scholar, 22Tsivkovskii R. Efremov R.G. Lutsenko S. J. Biol. Chem. 2003; 278: 13302-13308Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The results confirmed the important role of this residue for the P-type ATPase function and suggested that His-1069 may participate in the positioning of ATP within the catalytic site. However, direct evidence for the role of His-1069 in ATP binding has been lacking. Likewise, a number of other WD-causing mutations have been identified in the N-domain of WNDP, but their effects on nucleotide binding have not been characterized. Experiments using site-directed mutagenesis of the full-length P1-ATPases demonstrate that the dissection of the functional role of various residues could be impeded by additional effects of mutations on enzyme conformation (19Okkeri J. Bencomo E. Pietila M. Haltia T. Eur. J. Biochem. 2002; 269: 1579-1586Crossref PubMed Scopus (16) Google Scholar). The specific role of the amino acid residues in nucleotide coordination is particularly difficult to assess, because the nucleotide binding characteristics of the P1-type ATPase mutants have been analyzed indirectly by monitoring their ability to use ATP to form a phosphorylated intermediate or to transport copper. Phosphorylation and transport are several steps removed from the initial nucleotide-binding event, making conclusions regarding nucleotide binding tenuous. Therefore, to directly investigate the role of several residues in nucleotide binding by WNDP, we generated the recombinant WNDP N-domain and N-domain variants carrying known disease mutations, including H1069Q. In addition, we generated the recombinant N-domain of the Menkes disease protein (MNKP), the human copper-transporting ATPase ATP7A. MNKP is homologous to WNDP and is another member of the P1-ATPase subfamily. Comparison of these two proteins helped us to dissect the commonalities in their nucleotide-binding properties. Using direct ligand binding measurements, we demonstrate that 1) the N-domains of WNDP and MNKP contain a single nucleotide-binding site, 2) the properties of these sites are distinct from those of the P2-ATPases, and 3) the invariant residues Glu-1064 and His-1069 of WNDP are important for nucleotide coordination, whereas the less conserved Cys-1104 and Arg-1151 residues are not essential for nucleotide-binding. Expression Constructs for the Wild-type and Mutant N-domains of WNDP—To generate the expression constructs for wt N-domain of WNDP (amino acid residues Val-1036–Asp-1196 of the full-length protein), the corresponding cDNA region was amplified via PCR using pCDNA3.1(+)-WNDP plasmid as a template and the following primers: WT.ND-fwd, 5′-ACATATGGTCCCCAGGGTCATG-3′, and WT.ND-rev, 5′-AGAATTC TTAGTCTGCGATTGCGATC-3′. The PCR product, with 5′-NdeI and 3′-EcoRI endonuclease restriction sites flanking the coding sequence, was subcloned into the pCRII-Blunt TOPO vector (Invitrogen). The resulting pCRII-WT.ND plasmid was then used as a template for the generation of E1064A, H1069Q, C1104F, C1104A, and R1151H mutants by site-directed mutagenesis. The site-directed mutagenesis has been carried out in two PCR steps. In the first step, two overlapping PCR products were generated using pCRII-WT.ND as a template. Product A was produced with WT.ND-fwd primer and mutagenesis-rev primer; product B was generated using mutagenesis-fwd primer and WT.ND-rev primer. In the second PCR step, the overlapping products A and B were used as templates and were amplified using the WT.ND-fwd and WT.ND-rev primers, producing mutant N-domain. Mutagenesis primers were: E1064A.ND-fwd: 5′-GTGGTGGGGACTGCGGCCGCCAGCAGT-3′, E1064A.ND-rev: 5′-ACTGCTGGCGGCCGCAGTCCCCACCAC-3′, H1069Q.ND-fwd: 5′-AGCAGTGAACAA CCCTTGGGCGTGG-3′, H1069Q.ND-rev: 5′-ACGCCCAAGGGTTGTTCACTGCTGG-3′, C1104F.ND-fwd: 5′-TGTGGAATTGGGTTCAAAGTCAGCAAC-3′, C1104F.ND-rev: 5′-GTTGCTGACTTTGAACCCAATTCCACA-3′, C1104A.ND-fwd: 5′-TGTGGAATTGGGGC CAAAGTCAGCAAC-3′, C1104A.ND-rev: 5′-GTTGCTGACTTTGGCCCCAATTCCACA-3′, R1151H.ND-fwd: 5′-CTGATTGGAAACCACGAGTGGCTG-3′, and R1151H.ND-rev: 5′-CAGCCACTCGTGGTTTCCAATCAG-3′. The mutant products were subcloned into the pCRII vector. Then, using the NdeI and EcoRI restriction sites, the coding regions of the wt and mutant N-domains were excised from the pCRII vector and ligated into the IMPACT-CN expression vector pTYB12 (New England Biolabs). Automated DNA sequencing was used to verify the desired nucleotide sequence of the entire coding region for all pTYB12-ND plasmid constructs. The N-domain of MNKP—The expression construct for MNKP N-domain (residues Thr-1048–Asp-1230 of the full-length protein) was generated by PCR of the corresponding cDNA region of ATP7A using the following primers: MNKP-ND fwd, 5′-ATGAATGCTACCATTA CTCACGGAAC-3′, and MNKP-ND rev, 5′-ATGAATTCTTAGTCTGCAATGGCTATC-3′. The PCR product, containing 5′-BsmI and 3′-EcoRI endonuclease restriction sites flanking the coding sequence, was then cloned into the pTYB12 expression vector. The nucleotide sequence of the pTYB12-MNKP.ND was verified by automated DNA sequencing. Expression and Purification of the Recombinant N-domains—All N-domains were expressed as fusion proteins with a chitin-binding domain and an intein protein. In brief, Escherichia coli BL21 (DE3) cells were grown in ampicillin-supplemented (100 μg/ml) Luria-Bertani liquid media at 37° C until an A600 of ∼0.6 was reached. The expression was then induced by addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.5 mm, with shaking at 250 rpm for ∼12 h at 23° C. After harvest by centrifugation, cell pellets were re-suspended in 50 mm Tris, pH 8.2, and 500 mm NaCl, with complete EDTA-free protease inhibitor mixture (Roche) and disrupted by passing the suspension through a French press twice at 15,000 p.s.i. The lysate was centrifuged for 45 min at 15,000 rpm, and the soluble fraction was passed over a chitin resin. Purified resin-bound fusion protein was obtained after washing the chitin resin (New England Biolabs) with approximately 30 column volumes of 50 mm Tris, pH 8.2, and 500 mm NaCl. After the wash, the resin was equilibrated with the intein cleavage buffer containing 50 mm DTT, 50 mm Tris pH 8.2, and 150 mm NaCl. Cleavage buffer promotes intein-mediated excision of the N-domain from the fusion, allowing elution of non-tagged N-domain from the resin. The purified WNDP N-domains contained four non-native aminoterminal residues (AGHM) derived from the pTYB12 vector; the MNKP N-domain contained no non-native amino acids. Expression and purification of the MNKP N-domain was carried out as described for the WNDP N-domain, except that the induction with isopropyl-β-d-thiogalactopyranoside was carried out at 16 °C to improve solubility. Typical yields of wt and mutant N-domains ranged from 2–6 mg protein per liter of cell culture. CD Spectroscopy—Purified wt and mutant N-domains were dialyzed extensively against 50 mm NaH2PO4, pH 7.0, buffer and concentrated to 0.4 mg/ml using Amicon Ultra PL-10 centrifugal filters (Millipore) with a 10-kDa molecular mass cutoff. Concentrated samples were then centrifuged at 90,000 × g for 45 min to remove insoluble particles. Protein concentrations were initially determined spectrophotometrically using a theoretical extinction coefficient of 8850 m–1 cm–1 at 280 nm; subsequent amino acid analysis confirmed the spectrophotometrically determined concentrations. CD spectra were measured using an AVIV CD model 215 spectrometer; deconvolution of the spectra was carried out with the program CDDN1 (23Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2538) Google Scholar). The CD measurements were performed for three independent protein preparations of each mutant, and the resulting secondary structure values were averaged. Thermal denaturation was used to further compare folding of the wt and mutant N-domains. Temperature-dependent structural transitions, measured as molar ellipticity changes at 222 nm, were monitored from 25 to 80 °C using CD spectroscopy. Spectra were collected with the AVIV CD 215 spectrometer using a 0.1-cm rectangular CD cell. Molar ellipticity was monitored in 0.5-nm increments with 30-s equilibrations between measurements. Nucleotide Binding Measurements—Isothermal titration calorimetry (ITC) was used to analyze the nucleotide-binding properties of wt WNDP and MNKP N-domains and the E1064A, H1069Q, C1104A, and R1151H N-domain mutants. Purified proteins were dialyzed extensively against 50 mm NaH2PO4, pH 7.0, buffer and concentrated to 116 μm (∼2 mg/ml). Nucleotide solutions of 4 mm ATP disodium salt (Sigma), ATP monosodium salt (Acros Organics), AMP (Acros Organics), or GTP dilithium salt (Alexis Biochemicals) were prepared in the dialysis buffer immediately before each titration. For titrations with ATP-Mg2+ complex, 12 mm MgCl2 (Sigma) was included in the 4 mm ATP disodium salt solution. To ensure that the ITC data do not include enthalpy changes caused by hydrolysis of nucleotides by the N-domain, 1 mm ATP in 50 mm MES buffer, pH 6.0, was incubated with or without 1 mm N-domain at room temperature for up to 6 h. The amount of free Pi resulting from ATP hydrolysis (10 μm Pi, 1% of total ATP) was determined by EnzChek phosphate assay kit (Molecular Probes) and was found to be identical in the presence and absence of the N-domain. ITC experiments were performed with the VP-ITC titration calorimeter (Microcal, Inc.). Protein and nucleotide solutions were degassed by vacuum aspiration for 5 min before loading the samples into the ITC cell and syringe, respectively. All titrations were carried out at 25 °C, with a stirring speed of 300 r.p.m. and a 180-s duration between each 8-μl injection; thermal power was monitored every 8 s. Wild-type and mutant N-domains were titrated to saturation, where possible, with a 4 mm stock solution of nucleotide. Parallel experiments were performed by injecting nucleotide into buffer or buffer into N-domain to determine the heats of dilution. The heats of dilution were then subtracted from their respective N-domain nucleotide titrations before data analysis. Thermogram analysis was performed with the Origin 5.0 data analysis software provided with the VP-ITC instrument (OriginLab Corp). For each protein-nucleotide interaction, at least three titrations were performed on independently prepared N-domains. Titration thermograms were analyzed independently, and the obtained Kd values were averaged. To characterize binding of ATP using changes in the intrinsic tryptophan fluorescence, 11.5 μm WNDP N-domain in 50 mm NaH2PO4, pH 7.0, was titrated with increasing concentrations of ATP (0–3 mm) or the same volume of buffer. Using a model PTI-QM1 (Photon Technology International) fluorimeter, protein sample was excited at 295 nm (2-nm slit width), and fluorescence was monitored at an emission wavelength of 320 nm (1-nm slit width), which corresponds to the maximum emission wavelength for the N-domain. The final increase in protein sample volume caused by ATP addition did not exceed 1% and was accounted for in final calculation. Data were plotted as ratio of F/F0versus concentration of ATP. Molecular Modeling and Molecular Dynamics Simulations—A spatial model of the ATP-binding domain of WNDP (residues M996-R1322) was built via homology modeling based on the x-ray structure of the nucleotide-binding domain (residues Ala-320–Lys-758) of Ca2+-ATPase in the E1 ("open") state (PDB entry 1EUL (13Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar)). The details of the modeling experiments and validation criteria were described previously (24Efremov R.G. Kosinsky Y.A. Nolde D.E. Tsivkovskii R. Arseniev A.S. Lutsenko S. Biochem. J. 2004; (May 17, [Epub ahead of print])PubMed Google Scholar). In the current work, minor conformational changes were introduced into the loop 1062–1071 of the model using the following procedure. First, twenty models with different conformations of this loop were generated. Two models in which the side chains of Glu-1064 and His-1069 point toward the adenine binding cleft in the N-domain were selected for future studies. Both models were subjected to 5-ns molecular dynamics simulations in explicit water. The conformers extracted from the equilibrium parts of molecular dynamics trajectories were then employed in docking simulations with the ATP molecule as described previously (24Efremov R.G. Kosinsky Y.A. Nolde D.E. Tsivkovskii R. Arseniev A.S. Lutsenko S. Biochem. J. 2004; (May 17, [Epub ahead of print])PubMed Google Scholar). The molecular surfaces were mapped according to their hydrophobic properties using the molecular hydrophobicity potential approach (25Efremov R.G. Vergoten G. J. Phys. Chem. 1995; 99: 10658-10666Crossref Scopus (47) Google Scholar). The recombinant wt and mutant N-domains were expressed in E. coli and isolated after affinity chromatography on chitin beads and intein-mediated self-cleavage, as described under "Experimental Procedures." The expression levels, protein yields, and purities were similar for all N-domains. Folding and Nucleotide-binding Properties of the wt WNDP N-domain—Previous NMR studies of the N-domain of Na,K-ATPase, a P2-ATPase, revealed that the domain is folded and can bind ATP (26Hilge M. Siegal G. Vuister G.W. Guntert P. Gloor S.M. Abrahams J.P. Nat. Struct. Biol. 2003; 10: 468-474Crossref PubMed Scopus (93) Google Scholar). However, the apparent affinity of the N-domain for the nucleotides was extremely low (5–20 mm) compared with the high affinity of the full-length protein (0.3–1 μm). Likewise, the analysis of ATP binding by the proteolytic fragment of Ca2+-ATPase, containing the N-domain, yielded a high apparent Kd value of ∼0.7 mm (27Champeil P. Menguy T. Soulie S. Juul B. de Gracia A.G. Rusconi F. Falson P. Denoroy L. Henao F. le Maire M. Moller J.V. J. Biol. Chem. 1998; 273: 6619-6631Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), whereas characterization of the recombinant N-domain of Ca2+-ATPase produced a Kd value of ∼2.4 mm (28Abu-Abed M. Millet O. MacLennan D.H. Ikura M. Biochem. J. 2004; 379: 235-242Crossref PubMed Google Scholar). These results suggested that the formation of the high affinity site for ATP in P2-ATPases is a result of interaction between the N-domain and other regions of the proteins. Because the sequences of the P1- and P2-ATPase N-domains are dissimilar, we were interested in testing whether the isolated N-domain of WNDP, a P1-ATPase, can alone form a high affinity site for the nucleotide. Before nucleotide binding measurements, the folding of the recombinant wt N-domain of WNDP was examined. Analysis of the secondary structure of the N-domain using CD spectroscopy (Fig. 2) revealed that the N-domain of WNDP has the following secondary structure composition: ∼26% α-helix, ∼18.5% β-turn, and ∼22% β-sheet (Table I). Furthermore, thermal denaturation experiments demonstrated a sharp transition curve typical of a well folded soluble protein, with a Tm of ∼52 °C (Fig. 2, inset). Finally, the N-domain was found to be resistant to treatment with mild concentrations of trypsin (data not shown). All these results indicated that the recombinant N-domain of WNDP is well folded.Table IThe secondary structure composition of the wt WNDP N-domain and the WD mutantsWTE1064AH1069QC1104AR1151H%α-helix26 ± 226 ± 326 ± 325 ± 324 ± 3β-antiparallel11 ± 111 ± 112 ± 112 ± 112 ± 2β-parallel11 ± 111 ± 111 ± 111 ± 112 ± 1β-turn18 ± 119 ± 119 ± 119 ± 119 ± 1Other34 ± 233 ± 332 ± 333 ± 333 ± 3 Open table in a new tab The ability of the N-domain to bind nucleotides was examined using ITC. The N-domain does not contain the catalytic aspartate and is not expected to hydrolyze ATP. To verify this prediction and ensure that ITC measures only nucleotide-binding events and not hydrolysis of the nucleotides, we determined hydrolytic activity of the N-domain. The ATP hydrolysis in the presence of the N-domain did not exceed the spontaneous rate of ATP decay in the buffer even after prolonged (6 h) incubation. Thus, the enthalpy changes observed in the ITC experiments are caused by binding of the nucleotides to the N-domain. A representative thermogram for the calorimetric titration of wt N-domain with ATP is presented in Fig. 3A. The exothermic evolution of heat upon ATP injections, shown at the top, illustrates saturable nucleotide binding by the N-domain. Calculation of the enthalpy changes at various molar ratios of ATP to the N-domain (Fig. 3A, bottom) revealed that the best fit for the data corresponds to the presence of one ligand-binding site in the N-domain. In contrast, titration with GTP demonstrated no significant protein-nucleotide interactions. Enthalpy changes in this case were indistinguishable from the results of the nucleotide titration into buffer. Thus, the N-domain of ATP7B discriminates extremely well between adenosine and guanosine moieties, indicating selectivity for the nucleotides. Calculations of the apparent affinity for ATP yielded a Kd of 75.30 ± 3.62 μm, an unexpectedly low value, comparing with 0.7–5 mmKd values reported earlier for the N-domains of the P2-ATPases (26Hilge M. Siegal G. Vuister G.W. Guntert P. Gloor S.M. Abrahams J.P. Nat. Struct. Biol. 2003; 10: 468-474Crossref PubMed Scopus (93) Google Scholar, 27Champeil P. Menguy T. Soulie S. Juul B. de Gracia A.G. Rusconi F. Falson P. Denoroy L. Henao F. le Maire M. Moller J.V. J. Biol. Chem. 1998; 273: 6619-6631Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Abu-Abed M. Millet O. MacLennan D.H. Ikura M. Biochem. J. 2004; 379: 235-242Crossref PubMed Google Scholar). The ITC result was confirmed by measuring changes in the intrinsic tryptophan fluorescence of the N-domain upon addition of ATP (Fig. 3B), which yielded similar Kd value (110 ± 15 μm). This relatively high affinity enabled us to elucidate the
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