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• W2076577065 abstract "Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8Å resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking. Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8Å resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking. Copper is a meticulously regulated redox-active micronutrient found in a number of important enzymes, including cytochrome c oxidase and superoxide dismutase. Because free or excess intracellular copper can cause oxidative damage, both prokaryotes and eukaryotes have developed specific copper trafficking and transport pathways (1Rosenzweig A.C. Acc. Chem. Res. 2001; 34: 119-128Crossref PubMed Scopus (235) Google Scholar, 2Huffman D.L. O'Halloran T.V. Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (421) Google Scholar). Deficiencies in these processes are linked to human diseases, including Wilson and Menkes disease. In Wilson disease, accumulation of copper in the liver and brain leads to cirrhosis and neurodegeneration, and in Menkes disease, copper transport across the small intestine is impaired, leading to copper deficiency in peripheral tissues (3Llanos R.M. Mercer J.F.B. DNA Cell Biol. 2002; 21: 259-270Crossref PubMed Scopus (129) Google Scholar, 4Sarkar B. Chem. Rev. 1999; 99: 2535-2544Crossref PubMed Scopus (236) Google Scholar). Both disorders are caused by mutations in Cu+-transporting P1B-type ATPases (5Bull P.C. Cox D.W. Trends Genet. 1994; 10: 246-252Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 6Cox D.W. Moore S.D. J. Bioenerg. Biomembr. 2002; 34: 333-338Crossref PubMed Scopus (103) Google Scholar, 7Hsi G. Cox D.W. Human Genet. 2004; 114: 165-172Crossref PubMed Scopus (48) Google Scholar), enzymes that are found in most organisms and function in the cellular localization and/or export of cytosolic copper (8Argüello J.M. J. Membr. Biochem. 2003; 195: 93-108Crossref PubMed Scopus (219) Google Scholar, 9Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (754) Google Scholar). The Cu+-ATPases include eight transmembrane (TM) 4The abbreviations used are:TMtransmembraneBCAbicinchoninic acidC-MBDC-terminal CopA metal binding domainN-MBDN-terminal CopA metal binding domainCopZ-CTCopZ C-terminusCopZ-NTCopZ N-terminusN-MBDN-terminal CopA metal binding domainr.m.s.d.root mean square deviationDTTdithiothreitolMOPS4-morpholinepropanesulfonic acid. helices, of which three (TM6, TM7, and TM8) contribute invariant residues to form the transmembrane metal-binding site, a cytosolic ATP binding domain linking TM6 and TM7, an actuator domain between TM4 and TM5, and cytosolic metal binding domains (MBDs) of ∼60-70 amino acids that bind Cu+ (8Argüello J.M. J. Membr. Biochem. 2003; 195: 93-108Crossref PubMed Scopus (219) Google Scholar, 10Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). Whereas prokaryotic Cu+-ATPases typically have one or two MBDs, eukaryotic homologs have up to six such domains. Each MBD contains a highly conserved CXXC consensus sequence for binding Cu+ and adopts a βαββαβ fold (11Achila D. Banci L. Bertini I. Bunce J. Ciofi-Baffoni S. Huffman D.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5729-5734Crossref PubMed Scopus (137) Google Scholar, 12Banci L. Bertini I. Ciofi-Baffoni S. Gonnelli L. Su X.C. J. Biol. Chem. 2003; 278: 50506-50513Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Banci L. Bertini I. Ciofi-Baffoni S. Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2001; 276: 8415-8426Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 14Gitschier J. Moffat B. Reilly D. Wood W.I. Fairbrother W.J. Nat. Struct. Biol. 1998; 5: 47-54Crossref PubMed Scopus (212) Google Scholar) nearly identical to that of the Atx1-like cytosolic copper chaperones, including yeast Atx1, human Atox1, and bacterial CopZ (15Arnesano F. Banci L. Bertini I. Huffman D.L. O'Halloran T.V. Biochemistry. 2001; 40: 1528-1539Crossref PubMed Scopus (163) Google Scholar, 16Banci L. Bertini I. Conte R.D. Markey J. Ruiz-Dueñas F.J. Biochemistry. 2001; 40: 15660-15668Crossref PubMed Scopus (102) Google Scholar, 17Rosenzweig A.C. Huffman D.L. Hou M.Y. Wernimont A.K. Pufahl R.A. O'Halloran T.V. Structure (Lond.). 1999; 7: 605-617Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 18Wernimont A.K. Huffman D.L. Lamb A.L. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2000; 7: 766-771Crossref PubMed Scopus (355) Google Scholar, 19Wimmer R. Herrmann T. Solioz M. Wüthrich K. J. Biol. Chem. 1999; 274: 22597-22603Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). These chaperones also contain a CXXC motif and deliver Cu+ to one or all of the MBDs (20Cobine P.A. George G.N. Winzor D.J. Harrison M.D. Mogahaddas S. Dameron C.T. Biochemistry. 2000; 39: 6857-6863Crossref PubMed Scopus (48) Google Scholar, 21DiDonato M. Hsu H-F. Narindrasorasak S. Que Jr., L. Sarkar B. Biochemistry. 2000; 39: 1890-1896Crossref PubMed Scopus (106) Google Scholar, 22Forbes J.R. Hsi G. Cox D.W. J. Biol. Chem. 1999; 274: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Hamza I. Schaefer M. Klomp L.W.J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (241) Google Scholar, 24Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2000; 275: 18611-18614Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 25Walker J.M. Tsivkovskii R. Lutsenko S. J. Biol. Chem. 2002; 277: 27953-27959Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 26Wernimont A.K. Yatsunyk L.A. Rosenzweig A.C. J. Biol. Chem. 2004; 279: 12269-12276Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is not clear how Cu+ reaches the transmembrane metal-binding site and how the cytosolic chaperones participate in this process. transmembrane bicinchoninic acid C-terminal CopA metal binding domain N-terminal CopA metal binding domain CopZ C-terminus CopZ N-terminus N-terminal CopA metal binding domain root mean square deviation dithiothreitol 4-morpholinepropanesulfonic acid. The hyperthermophilic Cu+-ATPase CopA from Archaeoglobus fulgidus is readily expressed in fully active recombinant form, is highly stable, and contains all of the essential structural elements for copper transfer, including one N-terminal and one C-terminal MBD (27Mandal 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, 28Sazinsky M.H. Agarwal S. Argüello J.M. Rosenzweig A.C. Biochemistry. 2006; 45: 9949-9955Crossref PubMed Scopus (56) Google Scholar, 29Sazinsky 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). CopA is therefore an excellent model system both for investigating the mechanisms of P1B-type ATPases and for studying interactions between a cytosolic chaperone and its intact partner Cu+-ATPase. The only potential copper chaperone protein in the A. fulgidus genome, which we have designated A. fulgidus CopZ, differs from all other known copper chaperones in that it contains an additional 130 amino acids fused to the N terminus of a 60-residue CXXC-containing sequence that is homologous to Atx1-like chaperones and Cu+-ATPase MBDs (Fig. 1A). Notably, the A. fulgidus CopA C-terminal metal binding domain (CopA C-MBD) is the most similar to the CopZ C terminus, with 42% identity. The CopA N-terminal MBD (CopA N-MBD) is only 20% identical to CopZ. The novel N-terminal domain of CopZ (CopZ-NT) contains nine conserved cysteine residues and resembles uncharacterized 10-15-kDa proteins from other extremophilic Archaea (Fig. 1B). The A. fulgidus protein is the only one in which this domain is fused to a putative copper chaperone, however. In all the other extremophilic organisms that have a CopZ-NT homolog, the putative copper chaperone exists as a separate 70-amino acid protein, and its gene is not located in an operon with that encoding a CopZ-NT homolog, suggesting that their expression might not be linked. Here we describe the characterization and 1.8 Å resolution crystal structure of the A. fulgidus CopZ N terminus (CopZ-NT). Surprisingly, CopZ-NT contains a [2Fe-2S] cluster and a mononuclear zinc site. The fusion of a redox-active domain with a CXXC-containing copper chaperone is unprecedented and suggests previously unrecognized paradigms for copper trafficking and regulation. Cloning and Purification of CopZ and the CopA N-terminal MBD—The gene encoding CopZ (AF03456, GenBank™ accession number NP_069182) was cloned from A. fulgidus genomic DNA by PCR using the primers 5′-ATGATGCGATGCCCAGAATG-3′ and 5′-TCTCTTTCAAGCCGTGCAGA-3′. The purified gene and the plasmid pPRIBA1 (IBA, Germany) were digested with the restriction enzyme BsaI, purified, and ligated to create the plasmid pCOPZ, which fuses a 10-amino acid (SAWSHPQFEK) Strep-Tactin tag to the C terminus of the expressed gene product. The gene encoding CopZ was also cloned into pBAD/TOPO vector (Invitrogen) using the primers 5′-ATGATGCGATGCCCAGAATG-3′ and 5′-TCTCTTTTCAAGCCGTGCAGA-3′ to attach a His6 tag to the CopZ N terminus. The N-terminal domain of CopZ (residues 1-131, CopZ-NT) was PCR-amplified from the pCOPZ plasmid by using the primers 5′-CGGGAAGGTCTCTGCGCTTCCAACGGG-AAATCC-3′ and 5′-GCCCTTGGTCTCTAATGATCGATGCCCAGAAT-3′, which encode for a BsaI restriction site. As described above, the gene was inserted into the pPRIBA1 plasmid to create pCOPZNT. The C-terminal CXXC-containing copper chaperone domain (residues 132-204, CopZ-CT) was PCR-amplified from the pCOPZ plasmid to include the Strep-Tactin tag by using the primers 5′-GGAATTCCATATGGGTGAGAAGAAAGCGGCTAAAAG-3′ and 5′-CCGCTCGAGTTATTTTTCGAACTGCGGGTGGCTCCAAGC-3′, which incorporate 5′ NdeI and 3′ XhoI restriction sites. The purified gene product and a pET21b plasmid (Novagen) were digested, purified, and combined to create the pCOPZCT vector. The CopA N-MBD (residues 16-87) was cloned into a pASK-IBA3 vector after PCR amplification with the primers 5′-GCCCTTGGTCTCTAATGGAAAGAACCGTCAGAGTTAC-3′ and 5′-CGGGAAGGTCTCTGCGCTAGCAGCTTGCTCATCCACCACAC-3′ as described above to create the construct pCOPANT. BL21Star(DE3)pLysS Escherichia coli cells carrying the plasmid pSJS1240 encoding for rare tRNAs (tRNAArgAG(A/A)GG and tRNAIleAUA) were transformed with the pCOPZ and pCOPZNT and pCOPANT constructs. BL21(DE3)pLysS E. coli cells (Stratagene) were transformed with the pCOPZCT plasmid, and the His6-tagged CopZ construct was inserted into E. coli TOP10CP cells. All cell types were grown in Luria-Bertani media at 37 °C in the presence of 100 mg/liter carbenicillin and 20 mg/liter chloramphenicol. Media for cells harboring the pSJS1240 plasmid were supplemented with 70 mg/liter spectinomycin. At an A600 of ∼0.6-0.7, protein expression was induced by adding either 100-500 μm isopropyl β-d-thiogalactopyranoside to cells containing the pPRIBA1 and pET21 vectors, 200 μg/liter tetracycline to cells containing the pASK-IBA3 vector, or 0.02% arabinose to cells expressing His6-tagged CopZ from the pBAD/TOPO vector. For cells expressing CopZ or CopZNT, 100 μm ferrous ammonium sulfate was added to the media at induction and every hour thereafter. This addition helped to produce higher quantities of fully metal-loaded protein. The addition of 100 μm CuSO4 to the media yielded protein containing <0.1 equivalent of copper and did not appear to toxic to the cells as judged by the growth rate and quantity of cell paste. The cells were harvested by centrifugation at 6000 × g for 5 min 3-4 h after induction. The pellet was washed with 25 mm Tris-HCl, pH 7.0, 100 mm KCl, frozen in liquid nitrogen, and stored at -80 °C until further use. Full-length CopZ was also expressed as described above in cells grown in minimal media supplemented with 100 μm iron ammonium sulfate that contained less than 10 μm zinc. Streptactin-tagged CopZ, CopZ-NT, CopZ-CT, and the CopA N-MBD were purified by using a procedure identical to the one described for the A. fulgidus CopA ATP binding domain (29Sazinsky 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) except that 1 mm DTT was added to all of the buffers. The His6-tagged CopZ was purified on a nickel-nitrilo-triacetic acid column (Invitrogen) following the manufacturer's instructions. The purified protein was either exchanged into 20 mm MOPS, pH 7.0, 20 mm NaCl, 1 mm DTT, 5% glycerol by several concentration and dilution steps using an Amicon Ultra YM-10 or YM-5 concentrator or into 25 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm DTT by using a Sephadex G-25 column. The proteins were frozen at 30 mg/ml in liquid nitrogen and stored at -80 °C until further use. Protein concentrations were estimated by using the Bradford assay (Sigma). Site-directed Mutagenesis—Site-directed mutagenesis was performed by using the QuikChange method (Stratagene) and the pCOPZ vector. The DNA primers for 9 single Cys to Ser mutations in the N terminus are listed in Table 1. Mutations were verified by DNA sequencing. All CopZ mutants were expressed and purified from BL21Star(DE3)pLysS E. coli cells containing the pSJS1240 plasmid using the procedures described above.TABLE 1Oligonucleotide primers for site-directed mutagenesis of CopZC4S5′-ATGATGCGAAGCCCAGAATGCAGCACGGAAGC7S5′-GATGCCCAGAAAGCAGCACGGAAGGATGGAGC38S5′-GGATTTTTACTTCAGCTCTTTGGAGAGCTGCGAGGC43S5′-CTGCTCTTTGGAGAGCAGCGAGGTTGTTTACTTCC75S5′-CAAAGCCGGTTAGCTACTGCAACAGGGTTACAGAGC77S5′-CAAAGCCGGTTTGCTACAGCAACAGGGTTACAGAGC109S5′-CAGGAAAAGGAAAATGGAGCGTCGTTACCAACCCATCC118S5′-CATCCGGGAGAAGCTGCCACTGGCATCTGGC119S5′-CATCCGGGAGATGCAGCCACTGGCATCTGG Open table in a new tab Metal Binding Analysis—Apo-forms of the proteins were loaded with Cu+ by incubation with a 10 molar excess of CuCl2 or CuSO4 in 25 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm DTT, or 25 mm MOPS, pH 7.0, 25 mm NaCl, 5 mm DTT for 10-60 min at room temperature with gentle agitation. The unbound copper was removed by centrifuging in a 10-kDa cutoff Centricon Amicon-15 (Millipore, MA) after diluting the sample with 15-20 volumes of buffer without DTT or desalting over a PD-10 column (Bio-Rad). The amount of bound copper was determined by the BCA method (30Brenner A.J. Harris E.D. Anal. Biochem. 1995; 226: 80-84Crossref PubMed Scopus (169) Google Scholar). Briefly, the proteins were precipitated by mixing up to 55 μl of sample with 18.3 μl of 30% trichloroacetic acid. The pellet was separated by centrifugation for 5 min at 9,000 × g. The supernatant (66 μl) was mixed with 5 μl of 0.07% freshly prepared ascorbic acid and 29 μl of 2× BCA solution (0.012% BCA, 7.2% NaOH, 31.2% HEPES). After a 5-min incubation at room temperature, the absorbance at 359 and 562 nm was measured. CuCl2 solutions were used as standards. Concentrations of 2-10 μm Cu+ were within the linear range. The iron content was determined by using a ferrozine assay (31Stookey L.L. Anal. Chem. 1970; 42: 779-781Crossref Scopus (3594) Google Scholar), and acid-labile sulfide was quantified by using the method of Beinert (32Beinert H. Anal. Biochem. 1983; 131: 373-378Crossref PubMed Scopus (401) Google Scholar). Zinc content was determined by flame atomic absorption spectrometry and by ICP atomic emission spectrometry. The results of three measurements were averaged, and the concentration was determined from a standard curve. The presence of various metal ions was also investigated by using x-ray fluorescence spectroscopy at the sector 5 beamline at the Advanced Photon Source. A small sample of 2 mm CopZ in 25 mm Tris, pH 7.5, 100 mm NaCl, 5% glycerol was frozen at 100 K on a standard protein crystal mounting loop and exposed to x-rays tuned to the absorption edges of iron, cobalt, nickel, copper, zinc, and tungsten. Cu+ Transfer between CopZ and the CopA N-MBD—Apo-CopA N-MBD was incubated with Strep-Tactin resin in a column for 20 min at room temperature with gentle agitation. To separate unbound protein, the column was washed with 10 volumes of buffer W (25 mm Tris-HCl, pH 8.0, 150 mm NaCl, 10 mm ascorbic acid). His6-tagged CopZ loaded with 1.8 ± 0.1 Cu+ was added in 6.6-fold excess to the column containing bound CopA N-MBD and incubated for 10 min at room temperature to initiate copper exchange. The proteins were then separated by washing the column with 10 volumes of buffer W followed by elution of the CopA N-MBD with buffer W containing 2.5 mm 2-(4-hydroxyphenylazo)benzoic acid. Both the wash and elution fractions were collected and analyzed for copper and protein content. To confirm that only the CopA N-MBD was present in the elution fractions, each fraction was analyzed by SDS-PAGE. As a control, copper-loaded CopZ incubated with Strep-Tactin beads without bound CopA N-MBD was subjected to the procedure described above and demonstrated no copper loss. Strep-Tactin-bound apo-CopA N-MBD incubated with just buffer W did not acquire copper either. Reduction of Cu2+ by CopZ—Under anaerobic conditions, 1 mm CopZ and CopZ-NT in 25 mm MOPS, pH 7.0, 25 mm NaCl were reduced with 4-fold excess dithionite and desalted on a PD-10 column (Bio-Rad) equilibrated with 50 mm Tris, pH 7.0, 50 mm NaCl. A 10-fold excess of BCA and a 3-fold excess of CuSO4 were then added to the eluted protein and allowed to incubate for 4 h at 25 °C to detect the reduction of Cu2+ to Cu+ colorimetrically. As a control, 1 mm CopZ and CopZ-NT in 25 mm MOPS, pH 7.0, 25 mm NaCl were oxidized with 10 mm K3Fe(CN)6 under aerobic conditions, desalted, moved into the anaerobic chamber, and incubated with BCA and CuSO4 as described above. No color change was observed. X-ray Absorption Spectroscopy—XAS samples were prepared anaerobically and aerobically for as purified (oxidized) and dithionite-reduced CopZ and CopZ-NT. Multiple independent but reproducible samples that did not contain copper were prepared at 2.0-5.0 mm iron concentrations in 100 mm Tris, pH 8.0, 150 mm NaCl, 30% glycerol and transferred into Lucite sample cells wrapped with Kapton tape. Samples were immediately frozen in liquid nitrogen. Iron XAS data for full-length CopZ were collected at Brookhaven National Laboratory (NSLS) beamline X-9B using a Si(111) crystal monochromator equipped with a harmonic rejection mirror. Samples were kept at 24 K using a helium Displex cryostat, and protein fluorescence excitation spectra were collected using a 13-element germanium solid-state detector. Spectra were collected with a iron foil control in a manner described previously (33Cook J.D. Bencze K.Z. Jankovic A.D. Crater A.K. Busch C.N. Bradley P.B. Stemmler A.J. Spaller M.R. Stemmler T.L. Biochemistry. 2006; 45: 7767-7777Crossref PubMed Scopus (112) Google Scholar). During data collection, each spectrum was closely monitored for photoreduction. The data represent the average of 7-10 scans. XAS data were analyzed using the Macintosh OS X version of the EXAFSPAK program suite (available on line) integrated with the Feff version 7.2 software (35Ankudinov A.L. Rehr J.J. Phys. Rev. Lett. 1997; 56: R1712-R1715Google Scholar) for theoretical model generation. Processing methods and fitting parameters used during data analysis are described in detail elsewhere (33Cook J.D. Bencze K.Z. Jankovic A.D. Crater A.K. Busch C.N. Bradley P.B. Stemmler A.J. Spaller M.R. Stemmler T.L. Biochemistry. 2006; 45: 7767-7777Crossref PubMed Scopus (112) Google Scholar, 36Lieberman 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 (73) Google Scholar). Single scattering theoretical models were used during data simulation. Data were simulated over the spectral k range of 1 to 12.85 Å-1, corresponding to a spectral resolution of 0.13 Å (37Lee P.A. Citrin P.H. Eisenberger P. Kincaid B.M. Rev. Mod. Phys. 1981; 53: 769-806Crossref Scopus (1703) Google Scholar). When simulating empirical data, only the absorber-scatterer bond length (R) and Debye-Waller factor (σ2) were allowed to freely vary, whereas metal-ligand coordination numbers were fixed at quarter-integer values. The criteria for judging the best fit simulation and for adding ligand environments included a reduction in the mean square deviation between data and fit (F′) (38Riggs-Gelasco P.J. Stemmler T.L. Penner-Hahn J.E. Coord. Chem. Rev. 1995; 144: 245-286Crossref Scopus (99) Google Scholar), a value corrected for number of degrees of freedom in the fit, bond lengths outside the data resolution, and all Debye-Waller factors having values less than 0.006 Å2. EPR Spectroscopy—Dithionite-reduced and as-isolated 2 mm CopZ and CopZ-NT samples in 100 mm Tris, pH 7.0-10.0, 150 mm NaCl, 20-30% glycerol were frozen in liquid nitrogen in 3-mm inner diameter quartz EPR tubes. Cryoreduction was achieved by γ-irradiation of the samples by exposure to a 60Co source at a dose rate of 0.46 megarad h-1 for 5-10 min. Cryogenically reduced samples were annealed in cooled isopentane at various times and temperatures before being rapidly cooled to 77 K. X-band EPR spectra were recorded between 2 and 20 K on Bruker ESP300 or EMX spectrometers equipped with an Oxford Instrument ESR900 liquid helium cryostat. Structure Determination of the CopZ N Terminus—CopZ-NT was crystallized in a Coy anaerobic chamber at room temperature by using the sitting drop vapor diffusion method. Equal volumes of protein at ∼15 mg/ml in 20 mm MOPS, pH 7.0, 20 mm NaCl, 5% glycerol, 1 mm DTT were combined with a crystallization buffer comprising 100 mm sodium acetate, pH 4.6, 200 mm ammonium sulfate, 15-20% PEG 2000 MME. Dark red crystals grew within 2 days. The crystals were flash-frozen aerobically in a cryosolution consisting of 75 mm sodium acetate, pH 4.6, 100 mm ammonium sulfate, 20% PEG 2000 MME, 20% glycerol. Native and iron anomalous data were collected at 100 K to 2.3-1.8 Å resolution at the Advanced Photon Source on the sector 19 and 23 beamlines (Table 2).TABLE 2Data collection, phasing, and refinement statisticsData collectionIron peakNativeAPS beamlineGM/CA-CATSBC-CAT(sector 23)(sector 19)Wavelength (Å)1.740.979Resolution (Å)aValues in parentheses are for the highest resolution shell (1.84 to 1.78 Å).40.0-2.350.0-1.78Unique observations13,94829,750Total observations195,626194,503Completeness (%)100 (100)98.9 (93.3)Redundancy14.0 (14.0)6.5 (4.6)I/σ19.4 (19.3)13.0 (3.2)RsymbRsym = ∑i ∑hkl|Ii(hkl) - 〈I(hkl)〉|/∑hkl〈I(hkl)〉, where Ii(hkl) is the ith measured diffraction intensity, and 〈I(hkl)〉 is the mean of the intensity for the Miller index (hkl). (%)6.8 (16.8)6.3 (45.1)Iron sites used for phasing4Figure of merit (after density modification)0.374 (0.897)RefinementRwork (%)cRwork = ∑hkl||Fo(hkl)| - |Fc(hkl)||/∑hkl|Fo(hkl)|.20.9Rfree (%)dRfree = Rwork for a test set of reflections (5%).23.5Molecules per asymmetric units2No. of protein non-hydrogen atoms2066No. of protein non-hydrogen atoms157r.m.s.d. bond length (Å)0.0048r.m.s.d. bond angle (°)1.14Average B-value (Å2)37.7a Values in parentheses are for the highest resolution shell (1.84 to 1.78 Å).b Rsym = ∑i ∑hkl|Ii(hkl) - 〈I(hkl)〉|/∑hkl〈I(hkl)〉, where Ii(hkl) is the ith measured diffraction intensity, and 〈I(hkl)〉 is the mean of the intensity for the Miller index (hkl).c Rwork = ∑hkl||Fo(hkl)| - |Fc(hkl)||/∑hkl|Fo(hkl)|.d Rfree = Rwork for a test set of reflections (5%). Open table in a new tab After data collection, sections of the crystal exposed to the x-ray beam turned yellow, suggestive of photoreduction. The crystals belonged to the space group P212121 and had unit cell dimensions of a = 56.25, b = 64.50, c = 84.15. Data sets were indexed and scaled with HKL2000 (39Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar), and SOLVE (40Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) and CNS (41Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) were used to locate 4 iron atoms and calculate and refine phases to 2.3 Å resolution by the SAD method. After density modification, ARP/wARP was used for automatic model building (42Cohen S.X. Morris R.J. Fernandez F.J. Ben Jelloul M. Kakaris M. Parthasarathy V. Lamzin V.S. Kleywegt G.J. Perrakis A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2222-2229Crossref PubMed Scopus (152) Google Scholar). The remainder of the model was built with XtalView (43McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) and refined with CNS. Residues 1-130 were observed in one molecule in the asymmetric unit, and residues 2-130 were observed in the second molecule. A Ramachandran plot calculation with PROCHECK (44Laskowski R.A. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) indicated that 90% of the residues have the most favored geometry, and the rest occupy additionally allowed regions. The root mean square difference (r.m.s.d.) for backbone atoms between the two molecules in the asymmetric unit is 0.3 Å, and no significant structural differences were observed. Metal Content of CopZ—Purified CopZ and CopZ-NT are 23- and 14-kDa monomers, respectively, that have a distinct red color, whereas the 9-kDa CopZ-CT is colorless. The optical spectra of the full-length protein and CopZ-NT are identical with three absorption peaks at 340, 430, 480 nm and a shoulder at 550 nm (Fig. 2A). The spectra are most similar to those observed for [2Fe-2S]-containing proteins (45Messerschmidt A. Huber R. Wieghardt K. Poulos T. Handbook of Metalloproteins. John Wiley & Sons, Inc., New York2001Google Scholar). Features attributable to either a mononuclear iron center or a [4Fe-4S] cluster are not present. Upon reduction with dithionite, these spectral features disappear. Because the spectra of CopZ and CopZ-NT are identical, it is likely that the C terminus is not involved in assembly of the CopZ-NT metal centers. Consistent with a [2Fe-2S] cluster, both CopZ and CopZ-NT bound 1.7 ± 0.3 iron ions per protein molecule. Full-length CopZ contained ∼0.6 eq of zinc, and the isolated CopZ, CopZ-NT, and CopZ-CT did not contain copper. Only zinc and iron were detected by x-ray fluorescence spectroscopy. Copper Binding, Transfer, and Reduction—After incubation with excess CuSO4 and DTT and buffer exchange, CopZ, CopZ-NT, and CopZ-CT were determined to bind 2.1 ± 0.3, 1.4 ± 0.3, and 1.0 ± 0.4 Cu+ ions/protein, respectively. Thus, each domain binds a single Cu+ ion. Like all of the other Atx1-like proteins, CopZ-CT likely binds Cu+ via the conserved cysteines in the CXXC motif (see below). The presence of a Cu+ ion bound to CopZ-NT is unexpected. Copper transfer from His6-tagged CopZ to the CopA N-MBD was demonstrated by incubating Cu+-loaded chaperone with Strep-Tactin resin containing bound apo-CopA N-MBD, separating the individual proteins, and analyzing the copper content (Fig. 3). The CopA N-MBD was selected for these experiments because mutagenesis data indicate that the N-MBD, but not the C-MBD, is important for CopA activity (46Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref" @default.
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• W2076577065 title "Characterization and Structure of a Zn2+ and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus fulgidus" @default.
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