Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307941200
ISSN1083-351X
AutoresJonathan G. Heddle, David J. Scott, Satoru Unzai, Sam‐Yong Park, Jeremy R. H. Tame,
Tópico(s)Radioactive element chemistry and processing
ResumoBacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a “classical” periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be ∼10 μm and that for cobalt was approximately 20 times higher. Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a “classical” periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be ∼10 μm and that for cobalt was approximately 20 times higher. Nickel is a transition metal with appreciable affinity for oxygen, nitrogen, and sulfur. Its biological role in animals remains obscure, although it has been implicated in both metabolism and reproduction of mammals and birds (1.Anke M. Groppel B. Kronemann H. Grun M. IARC Sci. Publ. 1984; 53: 339-365Google Scholar, 2.Uthus E.O. Poellot R.A. Biol. Trace Elem. Res. 1996; 52: 23-35Crossref PubMed Scopus (28) Google Scholar). The biology of nickel is much better understood in bacteria. Its properties allow it to bind both DNA and proteins and disrupt many cellular functions when present in excess (3.Babich H. Stotzky G. Adv. Appl. Microbiol. 1983; 29: 195-265Crossref PubMed Scopus (68) Google Scholar). Several bacterial nickel efflux systems have been described that confer resistance to high levels of the metal (4.Grass G. Fan B. Rosen B.P. Lemke K. Schlegel H.-G. Rensing C. J. Bacteriol. 2001; 183: 2803-2807Crossref PubMed Scopus (79) Google Scholar, 5.Schmidt T. Schlegel H.-G. J. Bacteriol. 1994; 176: 7045-7054Crossref PubMed Google Scholar). At the same time, nickel is known to be an essential cofactor in several bacterial enzymes (6.Hausinger R.P. Microbiol. Rev. 1987; 51: 22-42Crossref PubMed Google Scholar) and is essential for anaerobic metabolism (7.Maroney M.J. Curr. Opin. Chem. Biol. 1999; 3: 188-199Crossref PubMed Scopus (106) Google Scholar), so the cell must be able both to import and export nickel to maintain an appropriate non-toxic concentration in the cytoplasm. Seven enzymes are known that depend on nickel (8.Watt R.K. Ludden P.W. Cell. Mol. Life Sci. 1999; 56: 604-625Crossref PubMed Scopus (104) Google Scholar), and some at least require metallochaperones for nickel incorporation. For example, the virulence of several pathogens including Helicobacter pylori is dependent on the nickel-containing enzyme urease (EC 3.5.1.5). A number of accessory proteins are required for the production of active urease including the nickel metallochaperone UreE (9.Remaut H. Safarov N. Ciurli S. van Beeumen J.J. J. Biol. Chem. 2001; 276: 49365-49370Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10.Song H.K. Mulrooney S.B. Huber R. Hausinger R.P. J. Biol. Chem. 2001; 276: 49359-49364Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Nickel incorporation in Escherichia coli hydrogenases also requires a GTP-binding accessory protein, HypB (11.Maier T. Jacobi A. Sauter M. Bock A. J. Bacteriol. 1993; 175: 630-635Crossref PubMed Google Scholar). The protein sequences of a number of microbial nickel uptake systems have been described (12.Eitinger T. Wolfram L. Degen O. Anthon C. J. Biol. Chem. 1997; 272: 17139-17144Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13.Wolfram L. Friedrich B. Eitinger T. J. Bacteriol. 1995; 177: 1840-1843Crossref PubMed Google Scholar, 14.Wolfram L. Bauerfeind P. J. Bacteriol. 2002; 184: 1438-1443Crossref PubMed Scopus (29) Google Scholar), but to date no structural information has been obtained. Nickel uptake by bacteria is of interest for bioremediation and water purification as well as its biological function and involvement in pathogenesis. The nickel transporter of E. coli encoded by the nik operon was originally discovered by the loss of hydrogenase activity in mutants generated using the MudI transposon (15.Wu L.F. Mandrand-Berthelot M.A. Biochemie. 1986; 68: 167-179Crossref PubMed Scopus (93) Google Scholar, 16.Wu L.F. Mandrand-Berthelot M.A. Waugh R. Edmonds C.J. Holt S.E. Boxer D.H. Mol. Microbiol. 1989; 3: 1709-1718Crossref PubMed Scopus (78) Google Scholar). The addition of 0.5 mm nickel led to the full recovery of hydrogenase activity, suggesting that the mutants were defective in nickel transport. This was later demonstrated directly in nickel transport experiments using 63Ni2+ (17.Navarro C. Wu L.F. Mandrand-Berthelot M.A. Mol. Microbiol. 1993; 9: 1181-1191Crossref PubMed Scopus (213) Google Scholar). Sequencing the nik locus showed five open reading frames, nikA-E-encoding proteins with significant similarity to the ABC-type dipeptide and oligopeptide import systems (18.Wu L.F. Navarro C. Mandrand-Berthelot M.A. Gene (Amst.). 1991; 107: 37-42Crossref PubMed Scopus (44) Google Scholar). ABC transporters in Gram-negative bacteria such as E. coli consist of three components: integral membrane proteins that create a pore through the inner membrane, membrane-associated ATP-hydrolyzing proteins, and a periplasmic binding protein (PBP). 1The abbreviations used are: PBPperiplasmic binding proteinDTTdithiothreitolPDBProtein Data Bank. These transporters allow the cell to import selectively the available nutrients and signaling peptides. An analysis of the E. coli genome suggests that it encodes 44 import systems and 13 export systems in the ABC family (19.Linton K.J. Higgins C.F. Mol. Microbiol. 1998; 28: 5-13Crossref PubMed Scopus (358) Google Scholar). The crystal structures of more than a dozen different periplasmic binding proteins have been determined and show a number of common features (20.Quiocho F.A. Ledvina P.S. Mol. Microbiol. 1996; 20: 17-25Crossref PubMed Scopus (456) Google Scholar). These proteins serve as the initial receptor for their respective ligands, which diffuse freely through the outer membrane of Gram-negative bacteria. In Gram-positive bacteria, these proteins are tethered to the cell by lipophilic tails (21.Sutcliffe I.C. Russell R.R. J. Bacteriol. 1995; 177: 1123-1128Crossref PubMed Scopus (332) Google Scholar). Periplasmic binding proteins vary in size from 25 to 59 kDa, the largest being the oligopeptide-binding protein OppA, which shows considerable sequence similarity to NikA. NikA is expressed as a pre-protein 524 amino acid residues long. The N-terminal 22 residue leader sequence directs translocation to the periplasm where it is removed to leave a 502 residue mature protein with a molecular mass of 56.3 kDa (17.Navarro C. Wu L.F. Mandrand-Berthelot M.A. Mol. Microbiol. 1993; 9: 1181-1191Crossref PubMed Scopus (213) Google Scholar). periplasmic binding protein dithiothreitol Protein Data Bank. Overall, solute-binding proteins show little sequence similarity but do show a number of conserved features. The two relatively rigid halves are connected by a hinge, which closes on substrate binding, completely enclosing the substrate in a manner often compared with a Venus fly-trap. The large conformational change has been confirmed for several solute-binding proteins by x-ray scattering in solution (22.Shilton B.H. Flocco M.M. Nilsson M. Mowbray S.L. J. Mol. Biol. 1996; 264: 350-363Crossref PubMed Scopus (111) Google Scholar). Until 1998, all of the known PBP structures could be classified into two groups depending on the topology of the connection between the two lobes (23.Fukami-Kobayashi K. Tateno Y. Nishikawa K. J. Mol. Biol. 1999; 286: 279-290Crossref PubMed Scopus (167) Google Scholar). The crystal structures of two metal binding proteins in the solute-binding protein family, however, show a very different connection between the two domains, forming a new family of metal binding receptors (24.Claverys J.-P. Res. Microbiol. 2001; 152: 231-243Crossref PubMed Scopus (135) Google Scholar). TroA is a zinc-binding protein from Treponema pallidum (25.Lee Y.-H. Dorwart M.R. Hazlett K.R.O. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. Nat. Struct. Biol. 1999; 6: 628-633Crossref PubMed Scopus (141) Google Scholar, 26.Lee Y.-H. Dorwart M.R. Hazlett K.R.O. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. J. Bacteriol. 2002; 184: 2300-2304Crossref PubMed Scopus (78) Google Scholar), and PsaA is a manganese/zinc-binding protein from Streptococcus pneumoniae (27.Lawrence M.C. Pilling P.A. Epa V.C. Berry A.M. Ogunniyi A.D. Paton J.C. Structure. 1998; 6: 1553-1561Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In these proteins, the polypeptide chain passes from one domain to the other only once, this connection being a long helix running most of the length of the protein. Unlike the β strands, which connect the lobes of most solute-binding proteins, the helix is rigid and permits only a very small conformational change on ligand binding. It has been suggested that this is necessary for tight metal binding given the small ligand size (26.Lee Y.-H. Dorwart M.R. Hazlett K.R.O. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. J. Bacteriol. 2002; 184: 2300-2304Crossref PubMed Scopus (78) Google Scholar). The crystal structure of a molybdate-binding protein from Azotobacter vinelandii, however, shows a more usual topology with a flexible hinge (28.Lawson D.M. Williams C.E.M. Mitchenall L.A. Pau R.N. Structure. 1998; 6: 1529-1539Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). OppA and DppA (the E. coli dipeptide-binding protein) are nearly twice the size of the smallest members of the PBP family, the extra residues forming a highly conserved domain, which is shared with NikA (29.Tame J.R.H. Murshudov G.N. Higgins C.F. Wilkinson A.J. Structure. 1995; 3: 1395-1406Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 30.Nickitenko A.V. Trakhanov S. Quiocho F.A. Biochemistry. 1995; 34: 16585-16595Crossref PubMed Scopus (84) Google Scholar). OppA is the most unselective PBP, being able to bind short peptides with relatively little side-chain preference (31.Tame J.R.H. Sleigh S.H. Wilkinson A.J. Ladbury J.E. Nat. Struct. Biol. 1996; 3: 998-1001Crossref PubMed Scopus (126) Google Scholar, 32.Davies T.G. Hubbard R.E. Tame J.R.H. Protein Sci. 1999; 8: 1432-1444Crossref PubMed Scopus (61) Google Scholar). In contrast, most solute-binding proteins are highly selective and show dissociation constants of around 1–0.1 μm for their ligands. Two previously published reports (33.de Pina K. Navarro C. McWalter W. Boxer D.H. Price N.C. Kelly S.M. Mandrand-Berthelot M.A. Wu L.F. Eur. J. Biochem. 1995; 227: 857-865Crossref PubMed Scopus (57) Google Scholar, 34.Salins L.L. Goldsmith E.S. Ensor C.M. Daunert S. Anal. Bioanal. Chem. 2002; 372: 174-180Crossref PubMed Scopus (45) Google Scholar) suggest very different values for the Kd for Ni2+ of NikA. We have solved the crystal structure of the protein in both liganded and unliganded forms and determined the binding constants for Ni2+ and Co2+ using titration calorimetry. Cloning, Expression, and Purification—The region of the nikA gene encoding mature NikA protein was cloned by PCR from E. coli (JM109) chromosomal DNA and inserted into the isopropyl-1-thio-β-d-galactopyranoside-inducible expression vector pET28b (Novagen) using NcoI and XhoI restriction sites. The nucleotide sequence of the gene was confirmed by DNA sequencing. In a suitable E. coli host strain, the resulting plasmid expresses mature NikA (with no histidine tag) in a folded soluble form in very high yield, up to 60 mg/liter culture. Without the leader peptide, the protein is not directed to the periplasm but accumulates in the cytoplasm. NikA is reported to bind to nickel-chelating columns with high affinity (33.de Pina K. Navarro C. McWalter W. Boxer D.H. Price N.C. Kelly S.M. Mandrand-Berthelot M.A. Wu L.F. Eur. J. Biochem. 1995; 227: 857-865Crossref PubMed Scopus (57) Google Scholar), but this method was not chosen for purification because of the difficulties often encountered in removing ligands from periplasmic binding proteins. Given the high yield, high solubility, and stability of the protein, purification of the nickel-free protein was achieved relatively easily. NikA was expressed in BL21(DE3) pLysS cells (Stratagene). Cell lysate in 50 mm Tris-Cl, pH 7.0, 2 mm EDTA, and 2 mm DTT was centrifuged at 34,000 rpm, 4 °C for 30 min, and the supernatant removed. Ammonium sulfate was added to a final concentration of 1 m, and the protein was then applied to a phenyl-Sepharose column (Amersham Biosciences) equilibrated in 50 mm Tris, pH 8.5, 2 mm EDTA, 2 mm DTT, and 1 m ammonium sulfate. NikA was eluted with a descending ammonium sulfate gradient and was found to elute at ∼500 mm ammonium sulfate. Fractions containing NikA were dialyzed into 50 mm Tris, pH 8.5, 2 mm EDTA, and 2 mm DTT and applied to a Q-Sepharose column (Amersham Biosciences) equilibrated with the same buffer. It was eluted with an ascending sodium chloride gradient and was found to elute at ∼100 mm NaCl. If further purification was necessary, NikA was passed down a Hiload 26/60 Superdex 200 gel filtration column (Pharmacia) at a flow rate of 1 ml/min. NikA was stored at 4 °C in 50 mm Tris, pH 8.5. NikA proved extremely soluble at pH >6.5 up to 190 mg/ml. This has allowed an almost complete assignment of the isotopically labeled 502 residue protein in NMR spectra using 1–2 mm samples. 2S. Rajesu, Y. Ito, J. Heddle, S. Unzai, S.-Y. Park, and J. R. H. Tame, manuscript in preparation. Selenomethionine-substituted protein was prepared using standard protocols (35.Hendrickson W.A. Horton J.R. LeMaster D.M. EMBO J. 1990; 9: 1665-1672Crossref PubMed Scopus (1008) Google Scholar). Crystallization—NikA was crystallized using the hanging drop method in both the presence and absence of nickel. Apo-NikA crystals were grown at 20 °C using 7.5 mg/ml NikA in 50 mm Tris-Cl, pH 8.5, and a 10-fold molar excess of nickel. The protein solution was mixed 1:1 with the mother liquor, 12.5% polyethylene glycol 4000, 0.3 m ammonium sulfate, and 50 mm sodium acetate, pH 5.0. Crystals grew as triangular prisms to a maximum length of 150 μm over 1–2 weeks. The selenium-substituted crystal was grown using 150 mg/ml protein in 50 mm Tris-Cl, pH 8.5 (no nickel), and a mother liquor containing 7.5% polyethylene glycol 4000, 0.3 m ammonium sulfate, and 50 mm sodium acetate, pH 5.0. These crystals were isomorphous with the apoNikA crystals grown with native protein. Similar conditions were used for the nickel-bound crystals. 15 mg/ml NikA in 50 mm Tris-Cl, pH 8.5, containing a 10-fold molar excess of nickel was mixed with an equal volume of the crystallization solution, 20% polyethylene glycol 2000, 0.3 m ammonium sulfate, and 100 mm sodium acetate, pH 5.5, and the same concentration of nickel chloride. Thin plates appeared within 1 week but took several months to thicken sufficiently for data collection. Apo-NikA crystallized in space-group P32 (two molecules per asymmetric unit) as triangular prisms, which diffracted to 1.85 Å. Multiwavelength data were collected to 2.5 Å from a selenomethionine-containing crystal at beamline BL44B2 at SPring8 (Harima, Japan). The anomalous signal from the selenium atoms was used to derive phases using SOLVE and RESOLVE programs (36.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar, 37.Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1755-1762Crossref PubMed Scopus (166) Google Scholar). Refinement—The apoNikA protein model was built using ARPwARP (38.Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2565) Google Scholar) and TURBO (39.Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1989Google Scholar) programs. REFMAC (40.Murshudov G.N. Vagain A.A. Lebedev A. Wilson K. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar) was used for refinement, and the CCP4 package (41.Collaborative Computational Project 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) was used for general data manipulation. The nickel-bound structure was solved by molecular replacement using AMoRE (42.Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar) with two separate halves of the nickel-free model cut at the hinge region. This provided clear solutions at both the rotation and translation steps in space group P21212. A complete molecule built from the two halves was then used as a search model in MOLREP (43.Vagin A.A. Teplyakov A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (690) Google Scholar) to find both complete molecules in the asymmetric unit. Data collection and refinement statistics are shown in Table I. Overall, the quality of the maps is excellent. An analysis of the structures was carried out with XTALVIEW (44.McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). Fitting regions of the refined structures to each other was carried out with the program FIT written by Dr. Guoguang Lu (Purdue University). The center of gravity and principal rotation axes of models were found using AMoRE (42.Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar). Coordinates and x-ray data have been deposited in PDB with entry codes 1uiu (unliganded) and 1uiv (liganded).Table IData collection and phasing statisticsSpace group/unit cell (Å)P32/a = b = 127.4, c = 60.7Wavelength (Å)0.9793 (peak)0.9797 (inflection)0.9824 (remote)Resolution range (Å)20.0-2.520.0-2.520.0-2.5Reflections (measured/unique)153,794/37,104153,162/36,987156,012/37,111Completeness (overall/outer shell) (%)aCompleteness and Rmerge are given for overall data and for the highest resolution shell. The highest resolution shells for the native, nickel complex, and multi-wavelength anomalous dispersion datasets are 1.92-1.85, 2.02-1.95, and 2.59-2.50 Å, respectively.97.6/84.297.1/80.497.6/84.0Rmerge (overall/outer shell) (%)aCompleteness and Rmerge are given for overall data and for the highest resolution shell. The highest resolution shells for the native, nickel complex, and multi-wavelength anomalous dispersion datasets are 1.92-1.85, 2.02-1.95, and 2.59-2.50 Å, respectively.8.5/18.97.8/18.97.2/19.1Redundancy (overall)4.14.14.2Mean 〈I/Σ (I)〉 (overall)6.96.76.9Phasing (20.0-2.5 Å)Mean figure of merit after RESOLVE phasing 0.51Refinement statisticsNativeNickel complexSpace group/unit cell (Å)P32/a = b = 126.9, c = 60.5P21212/a = 69.7, b = 192.8, c = 75.1Refinement resolution (Å)20.0-1.8520.0-1.95Reflections (measured/unique)206,476/81,622278,267/66,817Completeness (overall/outer shell) (%)aCompleteness and Rmerge are given for overall data and for the highest resolution shell. The highest resolution shells for the native, nickel complex, and multi-wavelength anomalous dispersion datasets are 1.92-1.85, 2.02-1.95, and 2.59-2.50 Å, respectively.91.1/78.694.6/72.1Rmerge (overall/outer shell) (%)aCompleteness and Rmerge are given for overall data and for the highest resolution shell. The highest resolution shells for the native, nickel complex, and multi-wavelength anomalous dispersion datasets are 1.92-1.85, 2.02-1.95, and 2.59-2.50 Å, respectively.,bRmerge = Σ Ii - 〈I〉/Σ Ii, where Ii is intensity of an observation and 〈I〉 in the mean value for that reflection. Summations are over all equivalents. R-factor = Σh||Fo(h) - Fc(h)||/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. The free R-factor was calculated with 5% of the data excluded from the refinement.4.5/38.44.1/11.4Redundancy (overall)2.54.2Mean 〈I/Σ(I)〉 (overall)17.516.7σ Cut-off/reflections used0.0/81,6220.0/66,817R-factorbRmerge = Σ Ii - 〈I〉/Σ Ii, where Ii is intensity of an observation and 〈I〉 in the mean value for that reflection. Summations are over all equivalents. R-factor = Σh||Fo(h) - Fc(h)||/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. The free R-factor was calculated with 5% of the data excluded from the refinement./free R-factor (%)bRmerge = Σ Ii - 〈I〉/Σ Ii, where Ii is intensity of an observation and 〈I〉 in the mean value for that reflection. Summations are over all equivalents. R-factor = Σh||Fo(h) - Fc(h)||/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. The free R-factor was calculated with 5% of the data excluded from the refinement.20.1/24.920.4/25.5Root mean square deviation bond lengths/bond angles (Å)0.023/2.00.023/2.1Water atoms553504Average B-factor (protein/water/Ni) (Å2)34/2926/30/35Ramachandran plot Residues in most favorable regions (%)89.491.7 Residues in additional allowed regions (%)10.07.8 Residues in generously allowed regions (%)0.60.5a Completeness and Rmerge are given for overall data and for the highest resolution shell. The highest resolution shells for the native, nickel complex, and multi-wavelength anomalous dispersion datasets are 1.92-1.85, 2.02-1.95, and 2.59-2.50 Å, respectively.b Rmerge = Σ Ii - 〈I〉/Σ Ii, where Ii is intensity of an observation and 〈I〉 in the mean value for that reflection. Summations are over all equivalents. R-factor = Σh||Fo(h) - Fc(h)||/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. The free R-factor was calculated with 5% of the data excluded from the refinement. Open table in a new tab Analytical Ultracentrifugation—Experiments were carried out using a Beckman XL-I instrument using a AnTi 60 rotor. All of the experiments were carried out at 20 °C. Sedimentation velocity data were collected at 40,000 rpm and analyzed with SEDFIT (45.Schuck P. Biophys. J. 1998; 75: 1503-1512Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) to yield plots of concentration versus sedimentation coefficient. Buffer density and protein partial molar volume were estimated using SEDNTERP (46.Laue T.M. Shah B. Ridgeway T.M. Pelletier S.L. Harding S.E. Horton J.C. Rowe A.J. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, United Kingdom1992: 90-125Google Scholar). Sedimentation equilibrium data were collected at 9, 12, and 16 krpm using six-channel centerpieces and three different concentrations of protein between A280 nm of 0.25 and 1.0. The buffer used was 0.1 m Tris-Cl, pH 7.5, and 0.1 m sodium chloride. Nickel chloride (where present) was used at a final concentration of 16 mm. Absorption and interference data were analyzed separately using the manufacturer's software. Isothermal Titration Calorimetry—Experiments were performed using a CSC 4200 instrument (Calorimetry Sciences Corporation). NikA was purified using a final gel filtration step with EDTA- and DTT-containing buffer to remove all of the traces of heavy metal ions. The protein was then extensively dialyzed against 50 mm HEPES, pH 7.0, and 100 mm sodium chloride. The NikA concentration used was typically 90 μm, and all of the experiments were carried out at 25 °C. 25 injections were made of 10 μl of the same buffer containing 1 mm nickel chloride, cobalt chloride, or calcium chloride. Blank runs with no protein present were used to measure the background dilution heats. Each experiment was repeated three times, and the results were analyzed with the manufacturer's software. Structure Determination—Crystallization of the protein was carried out using the hanging drop method in both the presence and absence of nickel. Nickel-free NikA crystallized in space group P32 as triangular prisms, which diffracted to 1.85 Å. Molecular replacement using the OppA model proved impossible, not surprisingly, given the variable extent to which the hinge may open. Phases were therefore derived by multi-wavelength anomalous dispersion using selenomethionine-substituted protein. A nearly complete model could be built automatically from the experimental phases after density modification. Excellent electron density was derived from the trigonal apoNikA crystals using the anomalous signal from 10 selenium atoms/monomer. Residues 1–3 at the N terminus and 500–502 at the C terminus are not visible in the final 1.85-Å electron density map. All of the residue numbers refer to the mature protein. In the presence of nickel, orthorhombic crystals were grown, which diffracted to 1.95 Å. Clear systematic absences were observed along the h00 and 0k0 axes, but the possibility of space group P212121 could not be ruled out completely from the inspection of the diffraction pattern alone. The open apoform of the protein showed clearly that the protein consists of two lobes, residues 4–245 and 471–499 forming lobe I and residues 246–470 forming a contiguous domain, lobe II. Molecular replacement using these separate lobes allowed the nickel-bound orthogonal crystal form to be solved very rapidly in space group P21212. On structure refinement, it became clear that a mutation had occurred at the surface residue Gln-361, which has been replaced by an arginine. Resequencing all of the clones from the original PCR showed that one of these clones has a single base mutation in this codon. Although the mutation is 30 Å from the nickel binding site, fresh wild-type protein was prepared for nickel binding studies. Isothermal titration calorimetry was carried out with both wild-type and mutant NikA. The crystal structures are of the mutant protein. Overall Shape—The closed nickel-bound form of NikA has a flattened tear shape highly similar to DppA and OppA. A DALI (47.Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1289) Google Scholar) search of PDB with apoNikA revealed high similarity to DppA (PDB code 1dpe; Z-score 34.3) and OppA (PDB code 1jev; Z-score 29.0) but much poorer scores for other structures. The overall structure of the protein is shown in Fig. 1. Of the 27 PDB structures found by DALI, only two others were PBPs, the lysine-arginine-ornithine-binding protein and molybdate-binding protein, with Z-scores of 3.2 and 2.2, respectively. Because there are two copies per symmetric unit for both crystal forms of NikA, there are four ways of matching a liganded and unliganded monomer. Least-squares fitting the 271 Cα atoms of lobe I in the apoform and bound form gave root mean square deviations between 0.56 and 0.99 Å. The largest shift is found for a small loop around Leu-172, which appears to be flexible. Subsequently, overlapping the 225 lobe II Cα atoms gave root mean square deviations between 0.60 and 0.75 Å. The rotation between lobes was between 12.2 and 17.5°, and the translation in each case was <0.3 Å. The conformational change is therefore very close to a pure rotation of one rigid lobe relative to another. A stereoview of the superimposed apostructure and nickel-bound structure is shown in Fig. 2. This rotation is quite modest compared with other PBPs but much larger than the 4° observed in the case of the zinc-binding protein TroA (25.Lee Y.-H. Dorwart M.R. Hazlett K.R.O. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. Nat. Struct. Biol. 1999; 6: 628-633Crossref PubMed Scopus (141) Google Scholar, 26.Lee Y.-H. Dorwart M.R. Hazlett K.R.O. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. J. Bacteriol. 2002; 184: 2300-2304Crossref PubMed Scopus (78) Google Scholar). The hinge motion in NikA is similar to those in OppA and DppA but smaller (30.Nickitenko A.V. Trakhanov S. Quiocho F.A. Biochemistry. 1995; 34: 16585-16595Crossref PubMed Scopus (84) Google Scholar, 48.Sleigh S.H. Tame J.R.H. Dodson E.J. Wilkinson A.J. Biochemistry. 1997; 36: 9747-9758Crossref PubMed Scopus (90) Google Scholar). OppA for example shows a hinge opening of 26° in the crystal structure of the open unliganded form (48.Sleigh S.H. Tame J.R.H. Dodson E.J. Wilkinson A.J. Biochemistry. 1997; 36: 9747-9758Crossref PubMed Scopus (90) Google Scholar). The apoNikA crystal structure suggests no reason why, in s
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