Solution Structures of a Cyanobacterial Metallochaperone

Artigo Acesso aberto Revisado por pares

Solution Structures of a Cyanobacterial Metallochaperone

2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês

10.1074/jbc.m402005200

ISSN

1083-351X

Autores

Lucia Banci, Ivano Bertini, Simone Ciofi‐Baffoni, Xun‐Cheng Su, Gilles P.M. Borrelly, Nigel J. Robinson,

Tópico(s)

Alzheimer's disease research and treatments

Resumo

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P1-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a βαββα fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1–0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. 2JNH couplings in the imidazole ring of His-61 establish coordination of Nϵ2 to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S2 complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes. The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P1-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a βαββα fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1–0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. 2JNH couplings in the imidazole ring of His-61 establish coordination of Nϵ2 to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S2 complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes. Most of the biosphere is either directly, or indirectly, reliant upon effective copper delivery to the thylakoids of primary producers. The conversion of light into useful chemical energy by cyanobacteria and plants involves the transfer of electrons within the thylakoid lumen between two membranous photosystems. Electrons are commonly transferred via plastocyanin-bound copper. Plastocyanin is located within the thylakoid lumen and is imported as an unfolded protein (1Bogsch E. Brink S. Robinson C. EMBO J. 1997; 16: 3851-3859Crossref PubMed Scopus (119) Google Scholar), necessitating a separate copper supply in order to form the holoenzyme.Analyses of mutants of the cyanobacterium Synechocystis PCC 6803 established that two copper-transporting P1-type ATPases, PacS and CtaA, plus a small soluble copper metallochaperone, Atx1 (herein referred to as ScAtx1), are required for normal photosynthetic electron transfer via plastocyanin and for the activity of a second thylakoid-located copper protein, a caa3-type cytochrome oxidase (2Tottey S. Rich P.R. Rondet S.A.M. Robinson N.J. J. Biol. Chem. 2001; 276: 19999-20004Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In common with related polypeptides from other bacteria, yeast, and man (4Banci L. Bertini I. Ciofi-Baffoni S. Del Conte R. Gonnelli L. Biochemistry. 2003; 42: 1939-1949Crossref PubMed Scopus (76) Google Scholar, 5Radford D.S. Kihlken M.A. Borrelly G.P.M. Harwood C.R. Le Brun N.E. Cavet J.S. FEMS Microbiol. Lett. 2003; 220: 105-112Crossref PubMed Scopus (80) Google Scholar, 6Wernimont A.K. Huffman D.L. Lamb A.L. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2000; 7: 766-771Crossref PubMed Scopus (347) Google Scholar, 7Arnesano F. Banci L. Bertini I. Cantini F. Ciofi-Baffoni S. Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2001; 276: 41365-41376Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), ScAtx1 directly interacts with soluble amino-terminal domains of P1-type copper ATPases (3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, unlike other copper metallochaperones, ScAtx1 from Synechocystis PCC 6803 associates with two such proteins, and there is a presumption that the vectors for copper transfer alternate in each of these two interactions. In Synechococcus PCC 7942, PacS is located in thylakoid membranes (8Kanamaru K. Kashiwagi S. Mizuno T. Mol. Microbiol. 1994; 13: 369-377Crossref PubMed Scopus (100) Google Scholar), whereas CtaA is thought to import copper at the plasma membrane (9Phung L.T. Ajlani G. Haselkorn R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9651-9654Crossref PubMed Scopus (102) Google Scholar). The phenotypes of ΔctaA and ΔpacS mutants of Synechocystis PCC 6803 are consistent with both ATPases transporting copper in an inward direction into the cytosol and then into the thylakoid lumen (3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This provides an attractive system for studying the process of copper transfer between a copper metallochaperone and its partners. In addition to PacS and CtaA, Synechocystis PCC 6803 also contains a P1-type ATPase, ZiaA, that transports zinc and not copper (10Thelwell C. Robinson N.J. Turner-Cavet J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10728-10733Crossref PubMed Scopus (152) Google Scholar) and has an amino-terminal domain with higher affinity for copper than for zinc (11Borrelly G.P.M. Rondet S.A.M. Tottey S. Robinson N.J. Mol. Microbiol. 2004; (in press)PubMed Google Scholar). A subdomain of ZiaA (ZiaAN) is predicted to form a ferredoxin-like fold analogous to the amino-terminal regions of the two copper transporters, but ScAtx1 gave no detectable two-hybrid interaction with this subdomain of ZiaA (11Borrelly G.P.M. Rondet S.A.M. Tottey S. Robinson N.J. Mol. Microbiol. 2004; (in press)PubMed Google Scholar) nor with the entire soluble amino-terminal region of ZiaA (3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Provided there is no freely available cytosolic copper in Synechocystis PCC 6803, as implied for Escherichia coli (12Changela A. Chen K. Xue Y. Holshen J. Outten C.E. O'Halloran T.V. Mondragon A. Science. 2003; 301: 1383-1387Crossref PubMed Scopus (493) Google Scholar), lack of an ScAtx1-ZiaAN interaction will impose a kinetic barrier to discourage formation of otherwise thermodynamically favored copper-ZiaAN complexes (11Borrelly G.P.M. Rondet S.A.M. Tottey S. Robinson N.J. Mol. Microbiol. 2004; (in press)PubMed Google Scholar) while copper traffics to the thylakoid. What are the surfaces of ScAtx1 that allow interaction with the copper transporters but not with ZiaA?There is considerable interest in understanding the submolecular processes by which copper is passed between partner proteins in cellular trafficking pathways to avoid intracellular metal release. NMR-derived solution structures revealed that loop 1, containing Cys ligands, and loop 5, containing Lys-65, of yeast Atx1 are dynamic during interaction with its cognate ATPase, apoCcc2. It has been proposed that the observed flexibility in loops 1 and 5 of yeast apoAtx1 may provide a trigger for copper release and allow the chaperone to adapt to its two partners, downstream of CTR1 and upstream of Ccc2 (7Arnesano F. Banci L. Bertini I. Cantini F. Ciofi-Baffoni S. Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2001; 276: 41365-41376Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Loop 1 of copper-CopZ from Bacillus subtilis assumes the conformation of aporather than copper-CopZ upon contact with the amino-terminal domain of CopA (4Banci L. Bertini I. Ciofi-Baffoni S. Del Conte R. Gonnelli L. Biochemistry. 2003; 42: 1939-1949Crossref PubMed Scopus (76) Google Scholar). This implies copper release from the metallochaperone to the exporter, and ΔcopZ mutants were subsequently shown to be copper-sensitive (5Radford D.S. Kihlken M.A. Borrelly G.P.M. Harwood C.R. Le Brun N.E. Cavet J.S. FEMS Microbiol. Lett. 2003; 220: 105-112Crossref PubMed Scopus (80) Google Scholar), consistent with a role in export rather than import. It is presumed that any analogous mechanism for ScAtx1 must be somehow adapted to encourage copper transfer to the metallochaperone from one P1-type ATPase as well as release to another. The copper coordination sphere of ScAtx1 was probed with EXAFS, 1The abbreviations used are: EXAFS, extended x-ray absorption fine structure; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; r.m.s.d., root mean square deviation; DTT, dithiothreitol; TOCSY, total correlation spectroscopy; REM, restrained energy minimization.1The abbreviations used are: EXAFS, extended x-ray absorption fine structure; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; r.m.s.d., root mean square deviation; DTT, dithiothreitol; TOCSY, total correlation spectroscopy; REM, restrained energy minimization. and 108 NOE-derived distance restraints in the region of presumptive loops 1 and 5 were used to analyze the spatial arrangement of residues in this region (13Borrelly G.P.M. Blindauer C.A. Schmid R. Butler C.S. Cooper C.E. Harvey I. Sadler P.J. Robinson N.J. Biochem. J. 2004; 378: 293-297Crossref PubMed Google Scholar). This supported copper binding to Cys-15 and Cys-12 from loop 1 plus His-61 from loop 5, with all NMR-refined models orientating these three residues at distances compatible with a trigonal copper (I) site. Conversion of His-61 to Arg altered two-hybrid interactions with PacS, but not with CtaA, implying that the interacting surfaces of the two ATPases are not identical in these regions (13Borrelly G.P.M. Blindauer C.A. Schmid R. Butler C.S. Cooper C.E. Harvey I. Sadler P.J. Robinson N.J. Biochem. J. 2004; 378: 293-297Crossref PubMed Google Scholar).Here we show copper liganding to Nϵ2 of His-61. The full structure of ScAtx1 is reported as a prelude to direct analyses of ScAtx1 complexes with PacS and CtaA and to identify features that account for noninteraction with the analogous amino-terminal domain of ZiaA. We establish that metal-loaded ScAtx1 forms dimers in vitro and that Cys-15 alone does not enter the copper site of the opposing monomer consistent with Cys-12 thiols and His-61 Nϵ2 commencing and/or concluding ligand exchange with PacS and CtaA. Flexibility in the region of loop 1 of apoScAtx1 is suggestive of Cys-12, rather than His-15, being the pioneer ligand during copper transfer from CtaA and allowing a model to be generated for the entire copper ligand sequence from the cell surface to the thylakoid.EXPERIMENTAL PROCEDURESScAtx1 Protein Expression and Purification—E. coli cells (BL21 (DE3)) were grown in Luria-Bertani or minimal media containing M9 salts. All buffers were prepared using Milli-Q deionized water. HEPES and Tris buffer salts were obtained from Melford Laboratories Ltd. and Roche Applied Science. Chromatography materials were purchased from Amersham Biosciences, and other reagents were obtained from Sigma. Plasmid pETATX1, for overexpression of ScAtx1, was generated as described previously (3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and recombinant protein produced in E. coli (BL21(DE3)) was exposed to copper (0.5 mm) in LB medium. Lysates (2.5 ml) were applied to Sephadex G-75 (2.5 × 50 cm), and fractions (5 ml) eluted in 25 mm Tris-HCl, pH 7.0, were analyzed for total protein and for copper by atomic absorption spectroscopy. Pooled copper peak fractions were applied to Q-Sepharose and sequentially eluted with 25 mm Tris-HCl, pH 7.0, followed by 0.7 m NaCl, 25 mm Tris-HCl, pH 7.0. Fractions were again analyzed for copper and protein, and copper-containing fractions were desalted on Sephadex G-25 in 25 mm Tris-HCl, pH 7.0. A single prominent band of the anticipated size was detected by PAGE, and the amino-terminal 10 residues of sequence (Beckman LF 3000 protein sequencer) previously confirmed the identity of this purified protein (3Tottey S. Rondet S.A. Borrelly G.P. Robinson P.J. Rich P.R. Robinson N.J. J. Biol. Chem. 2002; 277: 5490-5497Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 15N-Labeled ScAtx1 was prepared from cells grown in minimal medium containing M9 salts supplemented with 15NH4Cl and exposed to copper (0.02 mm).Purified polypeptide was incubated with 10 mm dithiothreitol, transferred to an N2 atmosphere chamber, and fractionated on Sephadex G-25 equilibrated in and eluted with hydrochloric acid, pH 2.0, with no added reductant. If pre-metalated protein was required, recovered material was exposed to an equivalent molar concentration of copper(I) chloride prior to the addition of 1 m HEPES, pH 7.0. (Some apo samples were prepared without addition of copper at this stage, but copper(I) was subsequently added to the final concentrated protein at pH 7.0.) Samples were further concentrated by passage through Q-Sepharose, sequentially washed with 25 mm HEPES, pH 7.0, followed by 0.7 m NaCl, 25 mm HEPES, pH 7.0. Fractions were again analyzed for protein, and selected fractions were desalted on Sephadex G-25, eluted with 50 mm sodium phosphate buffer, pH 7.0, 10% D2O. Copper content was finally checked through atomic absorption spectroscopy. Analytical gel filtration was performed on a Superdex 75 10/30 HR sizing column in 50 mm sodium phosphate buffer, pH 7, containing, when specified, 1.5 mm dithiothreitol. S. cerevisiae Ccc2a domain is available in our laboratory.NMR Experiments and Structure Calculations—NMR spectra were performed on Avance 800, 600, and 500 Bruker spectrometers operating at proton nominal frequencies of 800.13, 600.13, and 500.13 MHz, respectively. All the triple resonance (TXI 5-mm) probes used were equipped with pulsed field gradients along the z axis. The 500-MHz machine was equipped with a triple resonance cryoprobe.The NMR experiments were recorded on 15N-labeled and unlabeled samples. Two-dimensional TOCSY (14Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1192) Google Scholar) spectra were recorded on the 600-MHz spectrometers with a spin-lock time of 100 ms, a recycle time of 1.5 s, and a spectral window of 15 ppm. Two-dimensional NOESY maps (15Macura S. Wüthrich K. Ernst R.R. J. Magn. Reson. 1982; 47: 351-357Google Scholar, 16Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3517) Google Scholar) were acquired on the 800-MHz spectrometers with a mixing time of 100 ms, a recycle time of 1.5 s, and a spectral window of 15 ppm. The two-dimensional 15N-1H HSQC (17Palmer III, A.G. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar) maps and three-dimensional NOESY-15N HSQC experiments (100-ms mixing time) (18Kay L.E. Marion D. Bax A. J. Magn. Reson. 1989; 84: 72-84Google Scholar, 19Schleucher J. Schwendinger M. Sattler M. Schmidt P. Schedletzky O. Glaser S.J. Sorensen O.W. Griesinger C. J. Biomol. NMR. 1994; 4: 301-306Crossref PubMed Scopus (707) Google Scholar) were obtained at 800 MHz with an INEPT delay of 2.6 ms, a recycle time of 1.5 s, and spectral windows of 15 and 40 ppm for the 1H and 15N dimensions, respectively. HNHA experiments (20Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1046) Google Scholar) were performed at 800 or 500 MHz to determine 3JHNHα coupling constants. HNHB experiments (21Archer S.J. Ikura M. Torchia D.A. Bax A. J. Magn. Reson. 1991; 95: 636-641Google Scholar) were performed at 500 MHz. To identify the coordination mode of copper(I)-binding histidine, a 15N HSQC experiment was performed for measuring 2JNϵHδ, 2JNϵHϵ, 2JNδHϵ, and 3JNδHδ coupling constants (22Eijkelenboom A.P. Van den Ent F.M. Vos A. Doreleijers J.F. Hard K. Tullius T.D. Plasterk R.H. Kaptein R. Boelens R. Curr. Biol. 1997; 7: 739-746Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this experiment, the INEPT delay was set to 22 ms. All three-dimensional and two-dimensional spectra were collected at 298 K, processed with the standard Bruker software (XWINNMR), and analyzed with the XEASY program (23Eccles C. Güntert P. Billeter M. Wüthrich K. J. Biomol. NMR. 1991; 1: 111-130Crossref PubMed Scopus (269) Google Scholar).The backbone assignment has been performed using the three-dimensional 15N NOESY-HSQC, two-dimensional NOESY, and two-dimensional TOCSY spectra. Consequently, the combination of three-dimensional 15N NOESY-HSQC, HNHA, and HNHB and two-dimensional NOESY and TOCSY spectra allows the resonances assignment of side chains. Distance constraints for structure calculation were obtained from three-dimensional 15N NOESY-HSQC, two-dimensional NOESY experiments. 3JHNHα coupling constants, stemmed from HNHA experiments, were transformed through the Karplus equation into backbone dihedral ϕ angle constraints for structural calculations. The elements of secondary structure were determined on the basis of the 3JHNHα coupling constants and of the backbone NOEs from three-dimensional 15N NOESY-HSQC and two-dimensional NOESY spectra.An automated CANDID approach combined with DYANA torsion angle dynamics algorithm (24Herrmann T. Güntert P. Wüthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1321) Google Scholar) was used to assign the ambiguous NOE cross-peaks and to have a preliminary apo- and monomeric Cu(I)ScAtx1 structure. Structure calculations were then performed through iterative cycles of DYANA (25Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar) followed by restrained energy minimization (REM) with AMBER 5.0 (26Pearlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham T.E. Ferguson D.M. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER, Version 5.0. University of California, San Francisco1997Google Scholar) applied to each member of the family. Homodimeric Cu(I)ScAtx1 structure calculations were performed using the program DYANA (25Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar), and the 20 conformers with the lowest target function were minimized through AMBER 5.0 program package (26Pearlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham T.E. Ferguson D.M. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER, Version 5.0. University of California, San Francisco1997Google Scholar). The copper ion was included in the structure calculations of the copper(I)-loaded monomeric and dimeric forms following the same procedure already used for the monomeric and dimeric superoxide dismutase (27Banci L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Eur. J. Biochem. 2002; 269: 1905-1915Crossref PubMed Scopus (72) Google Scholar). The sulfur atoms of Cys-12 and Cys-15 and Nϵ2 of the imidazole ring of His-61 were linked to the metal ion through upper distance limits of 2.5 and 2.2 Å, respectively. This approach does not impose any fixed orientation of the ligands with respect to the copper(I) ion.The quality of the structures was evaluated using the programs PROCHECK-NMR (28Laskowski R.A. Rullmann J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4329) Google Scholar). The figure was generated with the program MOLMOL (29Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6469) Google Scholar). The atomic coordinates of apoScAtx1 have been deposited in the Protein Data Bank (code 1SB6).Relaxation Measurements and Analysis—Relaxation experiments were performed on Bruker Avance 600-MHz spectrometers at 298 K, and the protein sample concentrations range from 0.8 to 0.1 mm. 15N R1, R2, and steady-state heteronuclear NOEs were measured with pulse sequences as described by Farrow et al. (30Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1999) Google Scholar). R2 values were measured as a function of refocusing times (τCPMG) ranging from 450 to 1150 μs with the Carr-Purcell-Meiboom-Gill (CPMG) sequence (31Peng J.W. Wagner G. Methods Enzymol. 1994; 239: 563-596Crossref PubMed Scopus (249) Google Scholar, 32Mulder F.A. Van Tilborg P.J. Kaptein R. Boelens R. J. Biomol. NMR. 1999; 13: 275-288Crossref PubMed Scopus (71) Google Scholar) to identify backbone conformational exchange. In all experiments the water signal was suppressed with the “water flipback” scheme (33Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1007) Google Scholar). R1 and R2 experiments were acquired with 8 or 16 scans, whereas {1H}-15N NOE spectra were acquired with 48 scans. Duplication of measurements was performed to estimate the experimental uncertainty. A recycle delay of 3 s was used for R1 and R2 relaxation experiments except for the NOE experiments in which the recycle delay was 5 s. A total of 2048 K (1H) × 128 (15N) data points were collected. All spectra were processed with the XWINNMR program (Bruker) and analyzed with Sparky software (T. D. Goddard and D. G. Kneller, University of California, San Francisco).NMR relaxation data were fitted with the routine procedure implemented in the Sparky program. A search routine was used within Sparky to find the positions of the peak maxima. The exponential decay curves for R1 and R2 peak intensities were fitted to the two-parameter curve, h = A × exp(-R × t), where h is height and t is the variable delay parameter. The heteronuclear NOE values were obtained from the ratio of the peak intensity for 1H-saturated and unsaturated spectra. The error was estimated from 500 different fit trials, and peaks that had an error superior to 20% were not considered in the analysis. The experimental relaxation rates were used to map the spectral density function values, J(ωH), J(ωN), and J(0), following a procedure available in the literature (34Peng J.W. Wagner G. J. Magn. Reson. 1992; 98: 308-332Google Scholar).The overall rotational correlation time (τm) values were estimated from the R2/R1 ratio (35Brüschweiler R. Liao X. Wright P.E. Science. 1995; 268: 886-889Crossref PubMed Scopus (316) Google Scholar). The diffusion tensor of the molecule was estimated by fitting the local correlation times for the NH vector of each residue to an input structure with the program quadric_diffusion, available from the website of A. G. Palmer III (see Ref. 36Mandel M.A. Akke M. Palmer III, A.G. J. Mol. Biol. 1995; 246: 144-163Crossref PubMed Scopus (902) Google Scholar). In this analysis, care was taken to remove from the input relaxation data those NHs having an exchange contribution to the R2 value or exhibiting large amplitude internal motions on a time scale longer than a few hundred picoseconds, identified from low NOE values, as inclusion of these data would bias the calculated tensor parameters (37Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1781) Google Scholar, 38Tjandra N. Feller S.E. Pastor R.W. Bax A. J. Am. Chem. Soc. 1995; 117: 12562-12566Crossref Scopus (665) Google Scholar). The input structures for quadric_diffusion were the energyminimized average solution structures of monomeric apoScAtx1 and monomeric/dimeric Cu(I)ScAtx1.RESULTSSolution Structure and Mobility of apoScAtx1—The 15NHSQC spectrum of the apo form of ScAtx1 (64 amino acids) shows a good dispersion of the signals indicating that the protein is in a folded state. Assignments of the resonances of apoScAtx1 started from the analysis of the 1H-15N HSQC maps that allowed the identification of the 15N and 1HN resonances. Analysis of 15N-edited three-dimensional NOESY-HSQC and of two-dimensional NOESY and TOCSY maps allowed sequence-specific assignment. 57 of the expected 63 15N backbone amide resonances were observed and assigned. The backbone NH resonances are missing for residues Met-1, Thr-2, Ala-11, Cys-12, Glu-13, and Ala-14. Totally, the resonances of 95% of nitrogen atoms and 96% of protons were assigned. The 1H and 15N resonance assignments of the apo form are reported in supplemental Table 1.2100 NOE cross-peaks have been assigned and integrated, providing 1118 unique upper distance limits, of which 1017 are meaningful. 44 ϕ angles derived from a HNHA experiment were also used in structure calculations. After restrained energy minimization with AMBER program on each of 20 lowest target function structures, obtained from DYANA calculations, the root mean square deviation (r.m.s.d.) for protein backbone and heavy atoms to the mean structure (for residues 3–64) is 0.49 Å (with a variability of 0.12 Å) and 0.83 Å (with a variability of 0.14 Å), respectively. The penalties for distance constraints and angle constraints are 0.15 ± 0.06 Å2 and 0.02 ± 0.01 radians2, respectively. The statistical analyses of the REM family of apoScAtx1 structures are reported in Table I. The structure displays the following secondary structure elements: 2–7(β1), 16–27 (α1), 31–35 (β2), 39–45 (β3), and 47–60 (α2), in accordance with the 3JHNHα coupling constants, the dαN(i - 1,i)/dNα(i,i) ratios and the NOEs patterns. In Fig. 1, the 20 conformers of apoScAtx1 are shown as a tube, whose radius is proportional to the backbone r.m.s.d. of each residue.Table IStatistical quality analysis of the REM family and of the mean structure of apoScAtx1 from Synechocystis PCC 6803 REM means the energy minimized ensemble of 20 structures, 〈REM〉 is the energy minimized average structure of the ensemble.RSM violations per experimental distance constraint (Å)aThe number of experimental constraints for each class is reported in parenthesesREM (20 structures)〈REM〉Intraresidue (204)0.0148 ± 0.00210.0153Sequential (299)0.0080 ± 0.00170.0097Medium rangebMedium range distance constraints are those within residues (i,i + 2), (i,i + 3), (i,i + 4), and (i,i + 5) (241)0.0115 ± 0.00270.0151Long range (273)0.0134 ± 0.00190.0110Total (1017)0.0120 ± 0.00110.0125φ (44) (deg)00Average number of violations per structureIntraresidue4.65 ± 1.652Sequential2.75 ± 0.885Medium rangebMedium range distance constraints are those within residues (i,i + 2), (i,i + 3), (i,i + 4), and (i,i + 5)4.90 ± 1.43Long range6.70 ± 1.624Total19.00 ± 3.0014φ0.2 ± 0.050Average no. of NOE violations larger than 0.3 Å00Total NOE square deviations (Å2)0.15 ± 0.060.15Average torsion deviations (rad2)0.02 ± 0.010r.m.s.d. to the mean structure (3–64) (Å)0.49 ± 0.12 Å (BB)0.83 ± 0.14 (HA)Structural analysiscData resulted from the Ramachandran plot analysis over the assigned residues. In the PROCHECK statistics, the average hydrogen-bond energy within 2.5–4.0 kJ mol-1 and overall G-factor over –0.5 is expected to be a good quality structure % of residues in most favorable regions75.574.6 % of residues in allowed regions21.420.3 % of residues in generously allowed regions2.83.4 % of residues in disallowed regions1.01.3H-bond energy (kJ mol-1)3.63 ± 0.123.63Overall G-factor–0.22 ± 0.02–0.30a The number of experimental constraints for each class is reported in parenthesesb Medium range distance constraints are those within residues (i,i + 2), (i,i + 3), (i,i + 4), and (i,i + 5)c Data resulted from the Ramachandran plot analysis over the assigned residues. In the PROCHECK statistics, the average hydrogen-bond energy within 2.5–4.0 kJ mol-1 and overall G-factor over –0.5 is expected to be a good quality structure Open table in a new tab 15N R1, R2, and 1H-15N NOE values, which provide information on internal mobility, were measured at 600 MHz for all assigned backbone NH resonances. Such average values are 2.46 ± 0.07, 7.09 ± 0.19, and 0.80 ± 0.08 s-1, respectively. The experimental relaxation data are reported in the supplemental material (Fig. 1). The correlation time for molecular reorientation (τm), as estimated from the R2/R1 ratio, is 4.3 ± 0.3 ns, as expected for a protein of this

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Solution Structures of a Cyanobacterial Metallochaperone