Structure of the Two Transmembrane Cu+ Transport Sites of the Cu+-ATPases
2008; Elsevier BV; Volume: 283; Issue: 44 Linguagem: Inglês
10.1074/jbc.m803248200
ISSN1083-351X
AutoresManuel González‐Guerrero, Elif Eren, Swati Rawat, Timothy L. Stemmler, José Argüello,
Tópico(s)Enzyme function and inhibition
ResumoCu+-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu+ within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu+ transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu+. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu+-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu+ (Site I Ka = 1.3 fm–1, Site II Ka = 1.1 fm–1), in conjunction with reversible direct Cu+ transfer from chaperones, points to a transport mechanism where backward release of free Cu+ to the cytoplasm is largely prevented. Cu+-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu+ within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu+ transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu+. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu+-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu+ (Site I Ka = 1.3 fm–1, Site II Ka = 1.1 fm–1), in conjunction with reversible direct Cu+ transfer from chaperones, points to a transport mechanism where backward release of free Cu+ to the cytoplasm is largely prevented. Copper is an essential micronutrient (1Fraústro da Silva J.J.R. Williams R.J.P. The Biological Chemistry of the Elements. 2nd Ed. Oxford Univesity Press, New York2001: 418-435Google Scholar, 2Tapiero H. Townsend D.M. Tew K.D. Biomed. Pharmacother. 2003; 57: 386-398Crossref PubMed Scopus (761) Google Scholar). It has critical catalytic and electron transfer roles in a number of key proteins (tyrosinase, lysyl oxidase, ferroxidase ceruloplasmin, plastocyanin, etc.). However, when free, copper participates in the production of reactive oxygen species leading to cellular damage. Toward sustaining intracellular copper balance, transmembrane transport systems maintain the copper cell quota, Cu+ chaperone proteins traffic the bound metal to specific cellular targets, and metal-sensing transcription factors control copper dependent protein expression (3Lutsenko S. LeShane E.S. Shinde U. Arch. Biochem. Biophys. 2007; 463: 134-148Crossref PubMed Scopus (116) Google Scholar, 4Arnesano F. Banci L. Bertini I. Ciofi-Baffoni S. Molteni E. Huffman D.L. O'Halloran T.V. Gen. Res. 2002; 12: 255-271Crossref PubMed Scopus (235) Google Scholar, 5O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2000; 275: 25057-25060Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar). The metal coordination geometry in these proteins is central to the efficiency of the Cu+ mobilization processes. In this direction, the coordination should ensure the specificity and prevent the release of free Cu+ to the cytoplasm. Canonical copper metalloproteins have long been characterized and classified based on spectroscopic and magnetic properties (Types I, II, and III) (6Rosenzweig A.C. Sazinsky M.H. Curr. Opin. Struct. Biol. 2006; 16: 729-735Crossref PubMed Scopus (99) Google Scholar, 7MacPherson I. Murphy M. Cell Mol. Life Sci. 2007; 64: 2887-2899Crossref PubMed Scopus (116) Google Scholar, 8Sakurai T. Kataoka K. Cell Mol. Life Sci. 2007; 64: 2642-2656Crossref PubMed Scopus (118) Google Scholar). Their study has provided great detail on copper coordination in "permanent" sites where copper is bound during the functional life of the proteins. Cu+ linear coordination by invariant Cys residues of chaperone proteins has been described, providing insight into the mechanism of copper trafficking and exchange among similar domains (9Ralle M. Cooper M.J. Lutsenko S. Blackburn N.J. J. Am. Chem. Soc. 1998; 120: 13525-13526Crossref Scopus (59) Google Scholar, 10Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (603) Google Scholar). More recently, trigonal coordination by Cys2-His sites has been observed, for instance, in Mycobacterium tuberculosis transcription factor CsoR (11Liu T. Ramesh A. Ma Z. Ward S.K. Zhang L. George G.N. Talaat A.M. Sacchettini J.C. Giedroc D.P. Nat. Chem. Biol. 2007; 3: 60-68Crossref PubMed Scopus (261) Google Scholar). Alternatively, MetnHis was found in several Cu+-trafficking proteins located in the oxidizing periplasm of prokaryotes (12Bagai I. Liu W. Rensing C. Blackburn N.J. McEvoy M.M. J. Biol. Chem. 2007; 282: 35695-35702Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 13Loftin I.R. Franke S. Blackburn N.J. McEvoy M.M. Protein Sci. 2007; 16: 2287-2293Crossref PubMed Scopus (104) Google Scholar, 14Xue Y. Davis A.V. Balakrishnan G. Stasser J.P. Staehlin B.M. Focia P. Spiro T.G. Penner-Hahn J.E. O'Halloran T.V. Nat. Chem. Biol. 2008; 4: 107-109Crossref PubMed Scopus (197) Google Scholar). Despite this progress, Cu+ distribution and balance cannot be understood without describing the selective coordination during compartmental transmembrane transport. In eukaryotic cells, members of the Ctr family of proteins transport Cu+ inside the cell (15Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Ctr1 organizes as homotrimers forming transmembrane pores that facilitate Cu+ transmembrane translocation by an apparently energy-independent undefined mechanism (16Aller S.G. Unger V.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3627-3632Crossref PubMed Scopus (182) Google Scholar, 17Eisses J.F. Kaplan J.H. J. Biol. Chem. 2005; 280: 37159-37168Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although relevant Cu+-binding Met have been observed in the extracellular loops of Ctr1 (15Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar); none of the invariant transmembrane residues appear to be required for transport, and no direct coordination is evident (17Eisses J.F. Kaplan J.H. J. Biol. Chem. 2005; 280: 37159-37168Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). As a counterpart to influx systems, Cu+-ATPases are responsible for cytoplasmic Cu+ efflux. Mutations of the human Cu+-ATPase genes, ATP7A and ATP7B, lead to Menkes syndrome and Wilson disease, respectively (18Cox D.W. Moore S.D.P. J. Bioenerg. Biomembr. 2002; 34: 333-338Crossref PubMed Scopus (107) Google Scholar, 19Lutsenko S. Barnes N.L. Bartee M.Y. Dmitriev O.Y. Phys. Rev. 2007; 87: 1011-1046Crossref PubMed Scopus (605) Google Scholar). Cu+-ATPases are members of the superfamily of P-type ATPases (20Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (768) Google Scholar, 21Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (281) Google Scholar). These couple Cu+ transport to the hydrolysis of ATP, following a classical Post catalytic/transport cycle (19Lutsenko S. Barnes N.L. Bartee M.Y. Dmitriev O.Y. Phys. Rev. 2007; 87: 1011-1046Crossref PubMed Scopus (605) Google Scholar, 21Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (281) Google Scholar). In this mechanism, transmembrane metal-binding sites (TM-MBSs) 4The abbreviations used are: TM, transmembrane segment; MBS, metalbinding site; EXAFS, extended x-ray absorption fine structure; XANES, x-ray absorption near edge structure. are responsible for handling the ion during transmembrane translocation (22Mandal A.K. Yang Y. Kertesz T.M. Argüello J.M. J. Biol. Chem. 2004; 279: 54802-54807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These transmembrane sites are exposed to the cytoplasm when they receive the ion from the partnering Cu+ chaperone (23González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5992-5997Crossref PubMed Scopus (191) Google Scholar). Upon enzyme phosphorylation (formation of E1P intermediary), Cu+ is transiently occluded within the transmembrane region. The transported metal is released following the opening of the TM-MBS to the extracellular (vesicular/luminal) compartment (E2P intermediary). Because of the coupled nature of this mechanism, i.e. Cu+ transport is tied to ATP hydrolysis, the stoichiometry of transport and the associated energy requirements are linked to the number and structure of TM-MBSs. Cu+-ATPases consist of eight transmembrane segments (TM), two large cytosolic loops comprising the actuator, phosphorylation and nucleotide-binding domains, and regulatory metal-binding domains in their N terminus (exceptionally in the C terminus) (20Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (768) Google Scholar, 21Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (281) Google Scholar, 24Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (225) Google Scholar, 27Dmitriev O. Tsivkovskii R. Abildgaard F. Morgan C.T. Markley J.L. Lutsenko S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5302-5307Crossref PubMed Scopus (101) Google Scholar). Analysis of Cu+-ATPase TM sequences revealed the presence of only six invariant residues, two Cys in the sixth TM (H6), Asn and Tyr in the seventh TM (H7), and Met and Ser residues in the eighth TM (H8) (24Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (225) Google Scholar). Subsequent mutagenesis studies of Archaeoglobus fulgidus CopA, a model Cu+-ATPase, showed that these residues are required for completion of the Cu+-dependent steps in the ATPase catalytic/transport cycle (22Mandal A.K. Yang Y. Kertesz T.M. Argüello J.M. J. Biol. Chem. 2004; 279: 54802-54807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In this work, we describe the Cu+ coordination during transport by Cu+-ATPases. The invariant transmembrane amino acids form two Cu+-binding sites through trigonal coordination. The structures and characteristics of these sites support a model mechanism for the direct metal exchange with Cu+ chaperones while ensuring the unfeasibility of Cu+ ion release to the cytoplasm. Site-directed Mutagenesis and Protein Expression—Preparation of wild type CopA, C27A,C30A,C751A,C754A CopA (C2-CopA) and C27A,C30A,C380A,C382A,C751A,C754A CopA (C0-CopA) cDNAs cloned into the pCRT7/NT-TOPO/His vector (Invitrogen) have been described (28Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (90) Google Scholar). Mutations C380A, C382A, Y682A, N683A, M711A, and S715A were introduced in both C2-CopA and C0-CopA backgrounds using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutations were confirmed by DNA sequencing (MacrogenUSA, Rockville, MD). A streptavidin tag was introduced to the C terminus of CopA, C2-CopA, and C0-CopA by amplifying the coding cDNAs in the pCRT7/NT-TOPO/His vector (Invitrogen) using primer 5′-AGCGCTTGGAGCCACCCGCAGTTCGAAAAATAAAAGGGCGAATTCGAAGCTTGA and the complementary 3′ primer. The resulting amplicons were transformed into Escherichia coli BL21(DE3)pLysS cells (Invitrogen). Subsequently the His tags still encoded by these constructs were removed by amplification with primer 5′-TAAGACGATGACGATAAGGATAGGAGGCCAACCCTTATG and complementary 3′. These primers introduced a stop codon after the His coding sequence and placed a second ribosome-binding site (AGGAGG) prior to the ATG codon of the proteins. All of the constructs were introduced into BL21(DE3)pLysS E. coli cells (Invitrogen), and expression was induced with 0.75 mm isopropyl β-d-thiogalactopyranoside for 3 h. Enzyme Preparation—His-tagged proteins were prepared as previously described (29Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Streptavidin-tagged proteins were solubilized in a similar manner. After membrane solubilization, dodecyl-β-d-maltoside was removed by addition of Bio-Beads SM-2 (Bio-Rad), and protein purification was performed using streptavidin tag affinity chromatography as previously described (30Eren E. Kennedy D.C. Maroney M.J. Argüello J.M. J. Biol. Chem. 2006; 281: 33881-33891Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Purified proteins were stored in 50 mm HEPES, 200 mm NaCl, 10 mm ascorbic acid, pH 7.5, at –80 °C. Protein concentration determinations were performed in accordance to Bradford (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (221488) Google Scholar) using bovine serum albumin as a standard. The accuracy of colorimetric protein measurements was confirmed by total amino acid analysis (Keck Facility, Yale University, New Haven, CT). p-Nitrophenyl Phosphatase Assays—p-Nitrophenyl phosphatase activity determinations were performed as described (32Yang Y. Mandal A.K. Bredestón L.M. Luis González-Flecha F. Argüello J.M. Biochim. Biophys. Act. 2007; 1768: 495-501Crossref PubMed Scopus (26) Google Scholar). Determination of Cu+ Binding Stoichiometry and Affinity— Cu+ binding determinations were carried out in a buffer containing: 50 mm HEPES, pH 7.5, 200 mm NaCl, 0.01% dodecyl-β-d-maltoside, 0.01% asolectin, 10 mm ascorbic acid, 100 μm CuSO4, and 10 μm protein. When indicated 2 mm ATP, 0.2 mm Na3VO4, 0.1 mm AlF–4 or 0.2 mm LaCl3, and 2 mm ATP were included in the media together with 5 mm MgCl2. The system was allowed to reach equilibrium for 5 min at room temperature, and excess Cu+ was removed using a Sephadex-G25 column (Sigma). Following precipitation of proteins with 10% trichloroacetic acid, Cu+ was determined by BCA assay as described (33Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (174) Google Scholar). Cu+ binding affinities were determined by metal titrations of wild type or mutated proteins in the presence of BCA (23González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5992-5997Crossref PubMed Scopus (191) Google Scholar, 34Yatsunyk L.A. Rosenzweig A.C. J. Biol. Chem. 2007; 282: 8622-8631Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 35Deleted in proofGoogle Scholar). 10 μm protein and 25 μm BCA were titrated with 1–125 μm Cu+ in a buffer containing: 50 mm HEPES, pH 7.5, 200 mm NaCl, 0.01% dodecyl-β-d-maltoside, 0.01% asolectin, and 10 mm ascorbic acid, and the absorbance change between 330 nm and 410 nm was monitored. Protein absorbance was subtracted from the data. Free metal concentrations were calculated from KBCA = [BCA2Cu]/[BCAfree]2[Cu+], where KBCA is the association constant of BCA for Cu. An extinction coefficient of 43000 m–1 cm–1 at 359 nm for Cu+ bound BCA and KBCA of 4.6 × 1014 m–2 for Cu+ was used in determinations of free Cu+ concentration (23González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5992-5997Crossref PubMed Scopus (191) Google Scholar, 34Yatsunyk L.A. Rosenzweig A.C. J. Biol. Chem. 2007; 282: 8622-8631Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The Cu+-protein Ka and the number of metal-binding sites (n) were calculated from ν = n[Cu+]fKa/(1+Ka[Cu+]f), where ν is the molar ratio of Cu+ bound to protein. Reported errors for Ka and n are asymptotic standard errors provided by the fitting software (Origin, OriginLab, Northampton, MA). The plotted data points are the means ± S.E. of at least three experiments performed with independent protein preparations. XAS Analysis—Protein samples were fully loaded with Cu+ as described above and concentrated to 0.5 mm. XAS data were collected at the Stanford Synchrotron Radiation Laboratory on beamline 9-3, using a Si(220) double crystal monochromator equipped with a harmonic rejection mirror. The samples were maintained at 10 K using Oxford Instruments continuous-flow liquid helium cryostat. Protein fluorescence excitation spectra were collected using a 30-element Ge solid state array detector. A nickel filter (0.6 μm in width) and solar slits were placed between the cryostat and detector to filter scattering fluorescence not associated with protein copper signals. XAS spectra were measured using 5-eV steps in the pre-edge region (8750–8960 eV), 0.25 eV steps in the edge region (8986–9050 eV), and 0.05 Å–1 increments in the extended x-ray absorption fine structure (EXAFS) region (to k = 13.5 Å–1), integrating from 1 s to 20 s in a k3 weighted manner for a total scan length of ∼40 min. X-ray energies in the protein spectra were calibrated by collecting simultaneous copper foil absorption spectra, assigning the first inflection point in the foil spectra as 8980.3 eV. Every fluorescence channel of each scan was examined for spectral anomalies prior to averaging, and the spectra were closely monitored for photodamage. The data represent the averages of six to seven scans. XAS data were processed using the Macintosh OS X version of the EXAFSPAK program suite 5G. N. George, S. J. George, and I. J. Pickering, unpublished data. integrated with the Feff v7 software (36Ankudinov A.L. Rehr J.J. Phys. Rev. 1997; B56: R1712-R1715Crossref Scopus (534) Google Scholar) for theoretical model generation. Data reduction followed a previously published protocol for a spectral resolution in bond lengths of 0.13 Å (37Lieberman R.L. Kondapalli K.C. Shrestha D.B. Hakemian A.S. Smith S.M. Telser J. Kuzelka J. Gupta R. Borovik A.S. Lippard S.J. Hoffman B.M. Rosenzweig A.C. Stemmler T.L. Inorg. Chem. 2006; 45: 8372-8381Crossref PubMed Scopus (74) Google Scholar). EXAFS fitting analysis was performed on raw/unfiltered data. Protein EXAFS data were fit using single scattering Feff v7 theoretical models, calculated for carbon, oxygen, sulfur, and copper coordination to simulate copper-ligand environments, with values for the scale factors (Sc) and E0 calibrated by fitting crystallographically characterized copper model compounds, as previously outlined (37Lieberman R.L. Kondapalli K.C. Shrestha D.B. Hakemian A.S. Smith S.M. Telser J. Kuzelka J. Gupta R. Borovik A.S. Lippard S.J. Hoffman B.M. Rosenzweig A.C. Stemmler T.L. Inorg. Chem. 2006; 45: 8372-8381Crossref PubMed Scopus (74) Google Scholar). Criteria for judging the best fit EXAFS simulations utilized both the lowest mean square deviation between data and fit corrected for the number of degrees of freedom (F′) (38Kau L.S. Spira-Solomon D.J. Penner-Hahn J.E. Hodgson K.O. Solomon E.I. J. Am. Chem. Soc. 1987; 109: 6433-6442Crossref Scopus (989) Google Scholar) and reasonable Debye-Waller factors (σ2 < 0.006 Å2). Identification of Transmembrane Cu+-binding Sites in Cu+-ATPases—Characterization of Cu+-ATPase TM-MBSs required the isolation of a metal-bound form of the enzyme. Toward this goal, A. fulgidus CopA, a well characterized Cu+-ATPase, was obtained pure in a soluble (miscellar) largely monomeric form (21Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (281) Google Scholar, 22Mandal A.K. Yang Y. Kertesz T.M. Argüello J.M. J. Biol. Chem. 2004; 279: 54802-54807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Sazinsky M.H. Agarwal S. Argüello J.M. Rosenzweig A.C. Biochemistry. 2006; 45: 9949-9955Crossref PubMed Scopus (56) Google Scholar, 26Sazinsky M.H. Mandal A.K. Argüello J.M. Rosenzweig A.C. J. Biol. Chem. 2006; 281: 11161-11166Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (90) Google Scholar, 29Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 32Yang Y. Mandal A.K. Bredestón L.M. Luis González-Flecha F. Argüello J.M. Biochim. Biophys. Act. 2007; 1768: 495-501Crossref PubMed Scopus (26) Google Scholar, 39Cattoni D.I. González-Flecha F.L. Argüello J.M. Arch. Biochem. Biophys. 2008; 471: 198-206Crossref PubMed Scopus (18) Google Scholar). In initial experiments, the protein was incubated at room temperature in the presence of Cu+, and subsequently the free metal was removed by passage through a short Sephadex column. Four Cu+ ions were bound per CopA molecule (Table 1). Because CopA has two regulatory metal-binding domains able to bind Cu+ with high affinity, two of the bound Cu+ were associated with these domains (23González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5992-5997Crossref PubMed Scopus (191) Google Scholar). Metal binding to cytoplasmic metal-binding domains was removed by mutation of their Cu+-coordinating Cys. The resulting C27A,C30A,C751A,C754A CopA (C2-CopA) construct retained only the two Cys, Cys380 and Cys382, in H6. C2-CopA was able to bind two Cu+ per CopA molecule, suggesting the presence of two Cu+ sites in the transmembrane region (Table 1). A number of controls were performed to verify the association of these sites with transmembrane Cu+ transport. Streptavidin-tagged CopAs showed the same Cu+ binding as the initially tested His6-tagged constructs (Table 1). Identical results were obtained with proteins where the His6 tag was enzymatically cleaved. Determinations performed at high ionic strength (up to 1 m NaCl) yielded equal binding stoichiometry supporting a specific, high affinity Cu+ binding. Following these findings, it was relevant to establish the association of the sites still present in C2-CopA with Cu+ translocation. Conclusive evidence was obtained from Cu+-binding determinations performed in the presence of various ATPase ligands. The described result indicated that CopA TM-MBSs bind Cu+ with a very small koff that allows E1·Cu2+ separation by a relatively slow passage through a Sephadex column. However, in the presence of Mg2+-ATP and Cu+, the enzyme turns over, even at room temperature (where the turnover of the thermophilic CopA is minimal), and the transported ions are only transiently bound (28Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (90) Google Scholar, 29Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). That is, under these conditions Cu+ bound to transport sites would not remain bound during the passage through the Sephadex column, whereas ions not participating in catalysis/transport would stay bound to the protein. As expected, in the presence of Mg2+-ATP the wild type CopA was eluted with two Cu+ bound (to the metal-binding domains), whereas C2-CopA was unable to retain the metal, indicating the likely participation of both transmembrane Cu+ sites in transport (Table 1). Cu+ binding measurements in the presence of VO–34 or AlF–4 provided further support to this observation. These two inhibitors lock P-type ATPases in states analogous to the E2P conformation where Cu+ TM-MBS should not be accessible (29Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 40Missiaen L. Wuytack F. De Smedt H. Vrolix M. Casteels R. Biochem. J. 1988; 253: 827-833Crossref PubMed Scopus (58) Google Scholar). As expected, in the presence of VO–34 or AlF–4, CopA bound two Cu+, and C2-CopA was unable to bind the metal (Table 1).TABLE 1Determination of Cu+ binding stoichiometry to wild type and modified CopA proteinsCu+ bound/protein molar ratio2 mm ATP0.2 mm VO4-30.1 mm AlF4-0.2 mm La+3-ATPCopAaProteins containing a His6 tag.4.00 ± 0.302.29 ± 0.202.07 ± 0.133.87 ± 0.14C2-CopaProteins containing a His6 tag.1.83 ± 0.210.00 ± 0.030.00 ± 0.010.24 ± 0.502.16 ± 0.53CopAbProteins containing a streptavidin tag.3.94 ± 0.60C2-CopAbProteins containing a streptavidin tag.2.21 ± 0.07a Proteins containing a His6 tag.b Proteins containing a streptavidin tag. Open table in a new tab Having established the catalytic binding of at least two Cu+ ions, it was relevant to test the possible presence of additional transport sites. These hypothetical sites might have a lower affinity leading to metal dissociation during the relatively slow passage through the Sephadex column. In the case of the Na+,K+-ATPase and the Ca2+-ATPase, because of the reversible nature of ion binding to these enzymes, the metal-bound (occluded) forms occurring during catalysis can only be isolated by locking the enzymes in non-turnover conditions (low temperature, presence of inhibitors, etc.) and fast separation of free and protein bound substrates (41Glynn I.M. Richards D.E. J. Physiol. 1982; 330: 17-43Crossref PubMed Scopus (116) Google Scholar, 43Esman M. Skou J.C. Biochem. Biophys. Res. Commun. 1985; 127: 857-863Crossref PubMed Scopus (62) Google Scholar). Although CopA is a thermophilic protein with very slow turnover at room temperature (28Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (90) Google Scholar, 29Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), stabilization of metal bound enzyme was ensured by measuring Cu+ binding in the presence of Lanthanum-ATP. This inhibitor of P-type ATPases appears to lock these enzymes in cation occluded conformations (42Moreira O.C. Ríos P.F. Barrabin H. Biochim. Biophys. Acta. 2005; 1708: 411-419Crossref PubMed Scopus (25) Google Scholar, 44Fujimori T. Jencks W.P. J. Biol. Chem. 1990; 265: 16262-16270Abstract Full Text PDF PubMed Google Scholar). Lanthanum-ATP is able to inhibit CopA with a Ki of 20 μm (supplemental "Materials and Methods" and supplemental Fig. S1). In the case of CopA, no additional Cu+ binding was detected in the presence of Lanthanum-ATP (Table 1), reinforcing the concept that only two Cu+ TM-MBSs are involved in transport. Cu+-ATPases contain six invariant amino acids in the transmembrane region: two Cys in H6, Tyr and Asn in H7, and Met and Ser in H8 (24Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (225) Google Scholar). Proteins carrying mutations in these residues are unable to perform Cu+-dependent partial reactions (22Mandal A.K. Yang Y. Kertesz T.M. Argüello J.M. J. Biol. Chem. 2004; 279: 54802-54807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To ascertain their participation in the observed Cu+ TM-MBSs, individual mutations of these residues were introduced into the C2-CopA background. Proper folding of the resulting proteins was verified by measuring their Cu+-independent p-nitrophenyl phosphatase activity (Table 2). Previous studies have shown this activity in A. fulgidus CopA (32Yang Y. Mandal A.K. Bredestón L.M. Luis González-Flecha F. Argüello J.M. Biochim. Biophys. Act. 2007; 1768: 495-501Crossref PubMed Scopus (26) Google Scholar). Furthermore, inhibition by Lanthanum-ATP and AlF–4 indicated the specificity of this assay. These proteins carrying mutations of conserved transmembrane residues were all able to bind one Cu+ per molecule (Table 2), suggesting the participation of the replaced side chain in one of the two Cu+ TM-MBSs. Combination of these mutations provided a better description of each site. Replacement of C380A and C382A within the C2-CopA background yielded a Cys-less CopA (C0-CopA). This protein was still able of bind one Cu+, as did the Y682A C0-CopA construct (Table 2). Alternatively, mutations N683A, M711A, and S715A in the C0-CopA background removed Cu+ binding. Explaining these results, a simple model can be postulated where Cys380, Cys382, and Tyr683 form one TM-MBS (Site I), whereas Asn683, Met711, and Ser715 constitute the other (Site II). Participation of both transmembrane cysteines in a single site was unexpected considering previous reports hinting distinct roles for each cysteine (45Lowe J. Vieyra A. Catty P. Guillain F. Mintz E. Cuillel M. J. Biol. Chem. 2004; 279: 25986-25994Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Toward further verifying the identity of both sites, mutants were constructed where replacement of each transmembrane cysteine was combined with single mutations in Site II (Table 2). Supporting the proposed model, none of these proteins lacking a coordinating ligand in each site was able to bind Cu+.TABLE 2Determination of p-nitrophenyl phosphatase activity and Cu+ binding stoichiometry to modified CopA proteinsCu+ bound/proteinp-Nitrophenyl phosphatase activityμmol/mg/minCopA4.00 ± 0.302.07 ± 0.43CopA + 0.2 mm La+3-ATP2.16 ± 0.53-0.05 ± 0.07CopA + 0.1 mm AlF4-0.24 ± 0.500.05 ± 0.07C2-CopA1.83 ± 0.211.75 ± 0.13C0-CopA0.97 ± 0.041.83 ± 0.22C380A C2-CopA1.10 ± 0.061.59 ± 0.17C382A C2-CopA1.23 ± 0.101.48 ± 0.12Y682A C2-CopA0.98 ± 0.131.45 ± 0.18N683A C2-CopA1.12 ± 0.021.48 ± 0.15M711A C2-CopA0.92 ± 0.061.55 ± 0.14S715A C2-CopA0.94 ± 0.301.55 ± 0.11Y682A C0-CopA1.25 ± 0.631.43 ± 0.19N683A C0-CopA0.00 ± 0.011.50 ± 0.07M711A C0-CopA0.00 ± 0.011.48 ± 0.10S715A C0-CopA0.08 ± 0.101.43 ± 0.16C380A,M711A C2-CopA0.00 ± 0.011.61 ± 0.28C380A,S715A C2-CopA0.00 ± 0.011.49 ± 0.01C382A,M711A C2-CopA0.00 ± 0.011.70 ± 0.47C382A,S715A C2-CopA0.00 ± 0.011.51 ± 0.27
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