Chaperone-mediated Cu+ Delivery to Cu+ Transport ATPases
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.016329
ISSN1083-351X
AutoresManuel González‐Guerrero, Deli Hong, José Argüello,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoCu+-ATPases drive the efflux of Cu+ from the cell cytoplasm. During their catalytic/transport cycle, cytoplasmic Cu+-chaperones deliver the metal to the two transmembrane metal-binding sites (TM-MBSs) responsible for Cu+ translocation. Here, using Archaeoglobus fulgidus Cu+-ATPase CopA and the C-terminal Cu+-chaperone domain of CopZ (Ct-CopZ), we describe the mechanism of Cu+ transfer to both TM-MBSs. In absence of other ligands, Ct-CopZ transfers Cu+ to wild-type CopA and to various CopA constructs lacking or having mutated cytoplasmic metal-binding domains, in a fashion consistent with occupancy of a single TM-MBS. Similar experiments performed in the presence of 2.5 mm ADP-Mg2+, stabilizing an E1·ADP, lead to full occupancy of both TM-MBSs. In both cases, the transfer is largely stoichiometric, i.e. equimolar amounts of Ct-CopZ·Cu+ saturated the TM-MBSs. Experiments performed with CopA mutants lacking either TM-MBS showed that both sites are loaded independently, and nucleotide binding does not affect their availability. The nucleotide-induced E2→E1 transition is structurally characterized by a large displacement of the A and N domains opening the cytoplasmic region of P-type ATPases. Then, it is apparent that, whereas the first Cu+-chaperone can bind an ATPase form available in the absence of ligands, the second requires the E1·nucleotide intermediary to interact and deliver the metal. Interestingly, independent of TM-MBS Cu+ loading, nucleotide binding also prevents the regulatory interaction of the N-terminal cytoplasmic metal-binding domain with the nucleotide binding domain. Cu+-ATPases drive the efflux of Cu+ from the cell cytoplasm. During their catalytic/transport cycle, cytoplasmic Cu+-chaperones deliver the metal to the two transmembrane metal-binding sites (TM-MBSs) responsible for Cu+ translocation. Here, using Archaeoglobus fulgidus Cu+-ATPase CopA and the C-terminal Cu+-chaperone domain of CopZ (Ct-CopZ), we describe the mechanism of Cu+ transfer to both TM-MBSs. In absence of other ligands, Ct-CopZ transfers Cu+ to wild-type CopA and to various CopA constructs lacking or having mutated cytoplasmic metal-binding domains, in a fashion consistent with occupancy of a single TM-MBS. Similar experiments performed in the presence of 2.5 mm ADP-Mg2+, stabilizing an E1·ADP, lead to full occupancy of both TM-MBSs. In both cases, the transfer is largely stoichiometric, i.e. equimolar amounts of Ct-CopZ·Cu+ saturated the TM-MBSs. Experiments performed with CopA mutants lacking either TM-MBS showed that both sites are loaded independently, and nucleotide binding does not affect their availability. The nucleotide-induced E2→E1 transition is structurally characterized by a large displacement of the A and N domains opening the cytoplasmic region of P-type ATPases. Then, it is apparent that, whereas the first Cu+-chaperone can bind an ATPase form available in the absence of ligands, the second requires the E1·nucleotide intermediary to interact and deliver the metal. Interestingly, independent of TM-MBS Cu+ loading, nucleotide binding also prevents the regulatory interaction of the N-terminal cytoplasmic metal-binding domain with the nucleotide binding domain. Copper is an essential micronutrient involved in many critical processes (1Fraústro da Silva J.J.R. Williams R.J.P. The Biological Chemistry of the Elements. 2nd Ed. Oxford University Press, New York2001: 418-435Google Scholar, 2Tapiero H. Townsend D.M. Tew K.D. Biomed. Pharmacother. 2003; 57: 386-398Crossref PubMed Scopus (694) Google Scholar). However, it can also cause cellular damage due to its ability to catalyze the production of free radicals. To maintain the cell integrity while ensuring an adequate supply of copper, organisms have developed a complex molecular machinery to mobilize the metal. This involves copper-dependent transcription factors, chelators that sequester the excess metal, Cu+-chaperones that shuttle it to target proteins, and transporters that facilitate Cu+ translocation across the membranes (3Lutsenko S. LeShane E.S. Shinde U. Arch. Biochem. Biophys. 2007; 463: 134-148Crossref PubMed Scopus (111) Google Scholar, 4Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (264) Google Scholar, 5Kim B.E. Nevitt T. Thiele D.J. Nat. Chem. Biol. 2008; 4: 176-185Crossref PubMed Scopus (910) Google Scholar, 6Maryon E.B. Molloy S.A. Zimnicka A.M. Kaplan J.H. BioMetals. 2007; 20: 355-364Crossref PubMed Scopus (62) Google Scholar, 7Bird A.J. Adv. Microb. Physiol. 2008; 53: 231-267Crossref PubMed Scopus (16) Google Scholar, 8Balamurugan K. Schaffner W. Biochim. Biophys. Acta. 2006; 1763: 737-746Crossref PubMed Scopus (183) Google Scholar). Although the presence of chelators and Cu+-chaperones ensures the absence of free copper in the cell, it also creates the need of singular transmembrane transporters. These, although having structures similar to alkali metal carriers and pumps, should possess distinct transport mechanisms that deal with protein-metal and protein-protein affinity constants rather than metal concentration gradients across membranes (4Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (264) Google Scholar, 6Maryon E.B. Molloy S.A. Zimnicka A.M. Kaplan J.H. BioMetals. 2007; 20: 355-364Crossref PubMed Scopus (62) Google Scholar, 9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 10Xiao Z. Loughlin F. George G.N. Howlett G.J. Wedd A.G. J. Am. Chem. Soc. 2004; 126: 3081-3090Crossref PubMed Scopus (220) Google Scholar). Consequently, metal transport characteristics such as rate, specificity, energy requirement, etc., should be evaluated in light of these alternative paradigms. Cu+-ATPases are responsible for cytoplasmic Cu+ efflux. They are members of the P-type ATPase family of ion transporters, which also includes the well characterized Ca2+-ATPases, Na+/K+-ATPases and H+-ATPases, among others (11Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (747) Google Scholar, 12Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (416) Google Scholar, 13Fagan M.J. Saier Jr., M.H. J. Mol. Evol. 1994; 38: 57-99Crossref PubMed Scopus (146) Google Scholar). As all P-type ATPases, Cu+-ATPases couple ion (Cu+) transport to the hydrolysis of ATP following the essential elements of a classic Albers-Post cycle (4Argüello J.M. Eren E. González-Guerrero M. 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Briefly, cytoplasmic Cu+ binding to two transmembrane metal-binding sites (TM-MBSs) 2The abbreviations used are: TM-MBStransmembrane metal-binding siteBCAbicinchoninic acidΔN,C-CopACopA lacking both MBDsC2-CopACopA where MBDs have been mutatedC2N-MBD-CopACopA where Cys in TM-MBSs and C-MBD have been mutatedC0-CopACys-less CopAA-domainactuator domainATP-BDATP-binding domainP-domainphosphorylation domainN-domainnucleotide domainMBDmetal-binding domainBP fractionbound protein fractionUP fractionunbound protein fraction. 2The abbreviations used are: TM-MBStransmembrane metal-binding siteBCAbicinchoninic acidΔN,C-CopACopA lacking both MBDsC2-CopACopA where MBDs have been mutatedC2N-MBD-CopACopA where Cys in TM-MBSs and C-MBD have been mutatedC0-CopACys-less CopAA-domainactuator domainATP-BDATP-binding domainP-domainphosphorylation domainN-domainnucleotide domainMBDmetal-binding domainBP fractionbound protein fractionUP fractionunbound protein fraction. is coupled to ATP hydrolysis and enzyme phosphorylation (E1P(Cu+)2). Subsequently, the enzyme undergoes a conformational change (to E2P) leading to TM-MBSs opening to the vesicular/extracellular compartment with the consequent metal release. Enzyme dephosphorylation allows the return to the E1 form with TM-MBSs facing the cytoplasm. It is relevant that the E2→E1 transition is accelerated by ATP (or ADP) acting with low affinity; i.e. a modulatory mode (4Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (264) Google Scholar, 15Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 16Jensen A.M. Sørensen T.L. Olesen C. Møller J.V. Nissen P. EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (164) Google Scholar, 17Toyoshima C. Inesi G. Annu. Rev. Biochem. 2004; 73: 269-292Crossref PubMed Scopus (294) Google Scholar, 18Glynn I.M. Soc. Gen. Physiol. Ser. 1984; 38: 33-48PubMed Google Scholar, 19Post R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar). transmembrane metal-binding site bicinchoninic acid CopA lacking both MBDs CopA where MBDs have been mutated CopA where Cys in TM-MBSs and C-MBD have been mutated Cys-less CopA actuator domain ATP-binding domain phosphorylation domain nucleotide domain metal-binding domain bound protein fraction unbound protein fraction. transmembrane metal-binding site bicinchoninic acid CopA lacking both MBDs CopA where MBDs have been mutated CopA where Cys in TM-MBSs and C-MBD have been mutated Cys-less CopA actuator domain ATP-binding domain phosphorylation domain nucleotide domain metal-binding domain bound protein fraction unbound protein fraction. Cu+-ATPases consist of eight transmembrane helices, two large cytosolic loops comprising the actuator (A) and the ATP-binding domain (ATP-BD), which includes the phosphorylation (P) and nucleotide (N) domains, and cytoplasmic metal-binding domains (MBDs) in the N terminus (4Argüello J.M. Eren E. González-Guerrero M. Biometals. 2007; 20: 233-248Crossref PubMed Scopus (264) Google Scholar, 11Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (747) Google Scholar, 20Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (219) Google Scholar, 21Sazinsky M.H. Agarwal S. Argüello J.M. Rosenzweig A.C. Biochemistry. 2006; 45: 9949-9955Crossref PubMed Scopus (56) Google Scholar, 22Sazinsky 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 (81) Google Scholar, 23Dmitriev O. Tsivkovskii R. Abildgaard F. Morgan C.T. Markley J.L. Lutsenko S. Proc. Natl. Acad. Sci. 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N-MBDs have a regulatory role controlling the transporter turnover rate (28Fan B. Rosen B.P. J. Biol. Chem. 2002; 277: 46987-46992Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (89) Google Scholar, 30Voskoboinik I. Strausak D. Greenough M. Brooks H. Petris M. Smith S. Mercer J.F. Camakaris J. J. Biol. Chem. 1999; 274: 22008-22012Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In addition, they are involved in the important sorting of Cu+-ATPases observed in eukaryotes (14Lutsenko S. Barnes N.L. Bartee M.Y. Dmitriev O.Y. Physiol. Rev. 2007; 87: 1011-1046Crossref PubMed Scopus (560) Google Scholar). Lutsenko and collaborators have shown that a protein construct containing the six N-MBDs present in the human ATP7B Cu+-ATPase interacts with the ATP-BD in a Cu+-dependent manner (31Tsivkovskii R. MacArthur B.C. Lutsenko S. J. Biol. Chem. 2001; 276: 2234-2242Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Recently obtained structures of Archaeoglobus fulgidus CopA show the physical proximity between its single N-MBD, the A-domain, and the ATP-BD (24Wu C.C. Rice W.J. Stokes D.L. Structure. 2008; 16: 976-985Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These two observations could provide a structural basis for the regulatory role of cytoplasmic MBDs via Cu+-dependent domain-domain interactions (28Fan B. Rosen B.P. J. Biol. Chem. 2002; 277: 46987-46992Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (89) Google Scholar, 30Voskoboinik I. Strausak D. Greenough M. Brooks H. Petris M. Smith S. Mercer J.F. Camakaris J. J. Biol. Chem. 1999; 274: 22008-22012Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 31Tsivkovskii R. MacArthur B.C. Lutsenko S. J. Biol. Chem. 2001; 276: 2234-2242Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 32Argüello J.M. González-Guerrero M. Structure. 2008; 16: 833-834Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Transmembrane helices H6, H7, and H8 contain six conserved amino acids that form two metal-binding sites responsible for transmembrane transport: TM-MBS-I (two Cys in H6 and a Tyr in H7) and TM-MBS-II (Asn in H7, Ser and Met in H8) (see Fig. 1) (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Both sites coordinate Cu+ in a trigonal planar geometry. Mutation of any of these residues removes binding to the corresponding site and precludes Cu+-dependent partial reactions (33Mandal 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). Cytoplasmic Cu+ accesses the TM-MBSs bound to specific Cu+-chaperones (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Consequently, a specific transient interaction between the metal-loaded chaperone and the transmembrane ATPase by which the metal is delivered to TM-MBS can be predicted. This interaction and the associated Cu+ transfer are independent of Cu+ binding to N-MBDs, because these are not required for ATPase activation by chaperone bound Cu+. Both TM-MBSs bind Cu+ with high affinity (KaI = 1.12 ± 0.25 fM−1, and KaII = 1.3 ± 0.22 fM−1) preventing its backward release to cytoplasm (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Thus, saturation of both sites is easily achieved during equilibrium Cu+ binding assays performed with purified micellar proteins and "free" Cu+ ion; i.e. in the absence of metal chaperones. However, chaperone-mediated Cu+ transfer to the TM-MBS under equilibrium conditions leads to occupancy of a single site (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). If, as previously shown (33Mandal 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), metal binding to both TM-MBS is required for enzyme turnover, how is then the second TM-MBS loaded? High resolution structures of the sarcoplasmic reticulum Ca2+-ATPase have shown the large movements and coordinated interaction of cytoplasmic A-domain and ATP-BD during the catalytic cycle in P-type ATPases. These catalytic events driven by nucleotide binding, phosphorylation and dephosphorylation, are critical for metal binding, transmembrane occlusion, and translocation (35Toyoshima C. Arch. Biochem. Biophys. 2008; 476: 3-11Crossref PubMed Scopus (179) Google Scholar, 36Olesen C. Picard M. Winther A.M. Gyrup C. Morth J.P. Oxvig C. Møller J.V. Nissen P. Nature. 2007; 450: 1036-1042Crossref PubMed Scopus (373) Google Scholar). Experiments using limited proteolysis of Thermotoga maritima Cu+-ATPase suggest that similar large domain rearrangements also occur in heavy metal ATPases during transport (37Hatori Y. Majima E. Tsuda T. Toyoshima C. J. Biol. Chem. 2007; 282: 25213-25221Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Could the ligand-driven conformational transitions be required for chaperone-mediated Cu+ loading of transport sites? Employing the simple and well characterized A. fulgidus Cu+-ATPase CopA, we have further explored the Cu+ transfer into TM-MBSs by the corresponding chaperone, CopZ. We have observed that the equilibria for either TM-MBS (Ct-CopZ·Cu+ + TM-MBDs → Ct-CopZ + TM-MBDs·Cu+) are fully displaced toward saturation of CopA TM-MBSs. However, nucleotide binding to the ATP-BD is required for metal occupancy of the second transport site. This not only affects the CopZ-mediated Cu+ transfer to CopA but also disrupts the independent interaction of the regulatory N-MBD with ATP-BD. The A. fulgidus CopA and CopZ constructs used in this work are listed in the supplemental Table S1. cDNA coding for the A-domain of CopA was amplified from the CopA cDNA by PCR using primers 5′-ATGGGGGAGGCCATAAAGAAGCTCGTA-3′ and 5′-GCCCATCGCGTCCTCGACCAGCTT-3′. This DNA was cloned into pBAD/TOPO vector, which introduces a C-terminal hexahistidine tag suitable for Ni2+ affinity purification, and transformed into Escherichia coli Top10 cells. S715A C2N-MBD-CopA was the result of the ligation of the DNA fragments obtained from the HindIII and BsmI digestion of S715A C2-CopA and Wt-CopA previously cloned in pCRT7/NT-TOPO vector (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). This construct was subsequently transformed in B127(DE3)pLysS E. coli cells (Invitrogen). Expression and purification of membrane and soluble proteins were carried out as described (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Mandal A.K. Cheung W.D. Argüello J.M. J. Biol. Chem. 2002; 277: 7201-7208Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 22Sazinsky 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 (81) Google Scholar, 38Sazinsky M.H. LeMoine B. Orofino M. Davydov R. Bencze K.Z. Stemmler T.L. Hoffman B.M. Argüello J.M. Rosenzweig A.C. J. Biol. Chem. 2007; 282: 25950-25959Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Protein determinations were performed in accordance with Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Cu+ transfers from Strep-tagged Ct-CopZ to different His-tagged CopA constructs were performed as previously described (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Cu+-loaded Ct-CopZ was obtained by incubating equimolar amounts of Ct-CopZ and CuSO4 in a buffer containing 25 mm HEPES, pH 8.0, 500 mm NaCl, 10 mm ascorbic acid, and 0.01% n-dodecyl-β-d-maltopyranoside, 0.01% asolectin (Buffer T) at room temperature for 5 min with gentle agitation. No free Cu+ was detected after passing the Cu+-loaded Ct-CopZ through a Sephadex G-25 column (Sigma) and measuring Cu+ levels with the bicinchoninic acid (BCA) assay (40Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (169) Google Scholar). Except when indicated, Cu+ transfer assays were carried out by incubating 5 μm of indicated CopA constructs with concentrations of Ct-CopZ·Cu+ corresponding to 1.5 times the number of Cu+-binding sites available in the CopA construct; for example, 15 μm Ct-CopZ·Cu+ in the case of C2-CopA (which has two Cu+-binding sites) or 7.5 μm Ct-CopZ·Cu+ for C0-CopA (which has a single Cu+ site remaining) (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Cu+ transfer was performed in buffer T. 2.5 mm ADP-MgCl2 was included in the transfer assay media when the effect of nucleotide was tested. Following incubation at room temperature for 5 min, 150 μl of Strep-Tactin resin (IBA) pre-equilibrated in buffer T was added to bind Ct-CopZ. Unbound protein was separated by centrifugation at 14,000 rpm for 2 min. Resin-bound proteins were washed with 1 ml of buffer T, followed by elution with 0.5 ml of 2.5 mm desthiobiotin in buffer T. Protein and Cu+ concentration in the unbound and bound protein fractions were determined. SDS-PAGE (15% acrylamide gel) was performed to verify that only CopA-derived proteins were present in the unbound protein fraction and that only Ct-CopZ was present in the elution fraction (supplemental Fig. S1). Controls were performed where Cu+-loaded Ct-CopZ or CopA constructs were individually subjected to the same procedures; i.e. lacking the partner protein. Previously reported results showed that during these assays no significant amount of Cu+ is released from CopZ (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Interactions among cytoplasmic domains of CopA were studied by assessing the co-purification of isolated domains by batch affinity chromatography. 20 μm His-tagged ATP-BD or A-domain and 40 μm Strep-tagged N-MBD were incubated in a buffer containing 25 mm Tris-HCl, pH 8.0, 250 mm NaCl, 50 mm sucrose, and 5 mm dithiothreitol (Buffer I) for 10 min at room temperature with gentle agitation. The effects of Cu+ and nucleotides on domain-domain interaction were investigated by using Cu+-loaded N-MBD or including 5 mm ADP-MgCl2 in the assay media. Cu+-loaded N-MBD was obtained by incubating the protein with a five molar excess of CuSO4 in 25 mm Tris-HCl, pH 7.5, 10 mm ascorbic acid for 10 min at room temperature with gentle agitation. Unbound Cu+ was removed by passing through a Sephadex G-25 column (Sigma). Efficient Cu+ loading was verified by measuring Cu+ content of the eluted protein using the BCA assay (40Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (169) Google Scholar). The Cu+-loaded N-MBDs was used immediately after removing unbound Cu+ to minimize Cu+ dissociation. 200 μm bathocuproindesidulfonic acid was added to the reaction when Cu+-free conditions were required. Samples were incubated with 20 μl of Ni2+-nitrilotriacetic acid resin (Qiagen) for 10 min at room temperature and centrifuged at 14,000 rpm for 5 min to collect the unbound proteins in the supernatant (UP fraction). The proteins bound to the resin were washed with 200 μl of 5 mm imidazole in Buffer I and 200 μl of 20 mm imidazole in Buffer I, followed by elution with 50 μl of 150 mm imidazole in Buffer I (BP fraction). Protein content in the UP and BP fractions was analyzed by SDS-PAGE using 15% acrylamide gels and visualized by Coomassie Brilliant Blue staining (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). Controls were performed where each protein domain was individually subjected to the same procedures; i.e. lacking the interacting partner. Activation of Cu+-ATPases by Cu+-chaperones is independent of the presence or functionality of the N-MBD (or C-MBD) (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Thus, chaperones deliver Cu+ directly to the TM-MBDs of Cu+-ATPases. However, Cu+ transfer experiments performed under equilibrium conditions using excess of Cu+-chaperone to Cu+-ATPase lacking MBDs (CopZ·Cu+:ΔN,C-CopA, 3:1 molar ratio) showed transfer to and occupancy of an apparently single Cu+ site, albeit there are two TM-MBSs (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). This observation could be explained by the requirement of either larger excess of chaperone·Cu+ or functional N-MBDs for full occupancy of transport sites. Alternatively, because the metal transfer occurs through protein-protein interaction (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar), the need of a specific enzyme conformation able to open both TM-MBSs to chaperone·Cu+ can be postulated. Testing these hypotheses, we measured Cu+ transfer from the Cu+-loaded C terminus domain of A. fulgidus CopZ to various A. fulgidus CopA constructs. Assays were performed under equilibrium (non-turnover) conditions using soluble (micellar) forms of CopA. Ct-CopZ contains the classic Cu+-chaperone structure but lacks the ferredoxin-like N-terminal domain present in A. fulgidus CopZ (38Sazinsky M.H. LeMoine B. Orofino M. Davydov R. Bencze K.Z. Stemmler T.L. Hoffman B.M. Argüello J.M. Rosenzweig A.C. J. Biol. Chem. 2007; 282: 25950-25959Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). CopA proteins included Wt-CopA, CopA lacking one or both MBDs (ΔN,C-CopA and ΔN-CopA), and CopA containing mutated MBDs unable to bind Cu+ (C2-CopA). Previously reported control assays showed no appreciable release of free Cu+ during this type of experiment (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Table 1 shows that, in agreement with our previous observations, in the absence of other enzyme ligand Ct-CopZ·Cu+ transferred one Cu+ to TM-MBSs present in ΔN,C-CopA and C2-CopA. Experiments performed with up to five times molar excess of Ct-CopZ·Cu+ yielded identical results (not shown). Further supporting the single occupancy of TM-MBS under similar conditions, ΔN-CopA accepted two Cu+ from Ct-CopZ·Cu+, one in the C-MBD and the other in one of the two available TM-MBSs. Wt-CopA showed only slightly higher values (Table 1). However, analysis of this result is complicated by the partial transfer (not stoichiometric) of Cu+ from Ct-CopZ to N-MBD. We have reported that equilibrium exchange experiments performed with six times excess CopZ·Cu+ to N-MBD yielded only 35% of N-MBD·Cu+ (38Sazinsky M.H. LeMoine B. Orofino M. Davydov R. Bencze K.Z. Stemmler T.L. Hoffman B.M. Argüello J.M. Rosenzweig A.C. J. Biol. Chem. 2007; 282: 25950-25959Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Control experiments performed with a full-length CopA construct where the only remaining Cu+ site was at the N-MBD (S715A C2N-MBD-CopA) showed a similar equilibrium with only 24–34% Cu+ loading of N-MBD in the presence of 1.5 excess Ct-CopZ·Cu+ (Table 1). On the contrary, CopZ·Cu+ fully transfers the metal to C-MBD (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Thus, it could be assumed that in the Wt-CopA one Cu+ occupies the C-MBD, a second is in a TM-MBS, and the N-MBD is partially occupied. Although these data indicated that the MBDs are indeed not necessary for Ct-CopZ·Cu+ delivery of Cu+ to CopA TM-MBDs, they do not provide an explanation for the apparent loading of a single TM-MBS. We realize that the described results could also be explained by 50% occupancy of each TM-MBS. However, experiments described below, using mutants where a single TM-MBS is available, show that this is not the case.TABLE 1Cu+ transfer to CopA by Ct-CopZConstructCu+ ions/CopA moleculeaValues are the mean ± S.E. (n = 3).No nucleotide2.5 mm ADP-MgCl2ΔN,C-CopA0.8 ± 0.1bDatum was published in Ref. (34).1.8 ± 0.2C2-CopA1.1 ± 0.21.9 ± 0.1ΔN-CopA1.9 ± 0.22.9 ± 0.3Wt-CopA2.2 ± 0.13.1 ± 0.1S715A C2N-MBD-CopA0.2 ± 0.20.3 ± 0.1N683A C0-CopA0.1 ± 0.00.0 ± 0.0ΔN-CopA/Ct-CopZ1:2 ratio1:3 ratio2.0 ± 0.12.8 ± 0.1a Values are the mean ± S.E. (n = 3).b Datum was published in Ref. (34González-Guerrero M. Argüello J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5992-5997Crossref PubMed Scopus (187) Google Scholar). Open table in a new tab Loading of TM-MBDs sites by "free" Cu+ has shown that both sites accept the metal in the absence of any ligand stabilizing a particular conformation (9González-Guerrero M. Eren E. Rawat S. Stemmler T.L. Argüello J.M. J. Biol. Chem. 2008; 283: 29753-29759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Despite this, a certain possibility was that the interaction of the second Ct-CopZ·Cu+ with CopA requires a different enzyme conformation. Testing this hypothesis, Cu+ transfer experiments were performed in the presence of 2.5 mm ADP-Mg2+. This drives the enzyme into an E1 conformation, but phosphorylation followed by Cu+ release cannot occur. Under this condit
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