Unique Features of the sodC-encoded Superoxide Dismutase from Mycobacterium tuberculosis, a Fully Functional Copper-containing Enzyme Lacking Zinc in the Active Site
2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês
10.1074/jbc.m404699200
ISSN1083-351X
AutoresLaura Spagnolo, I Törö, Melania D’Orazio, Peter O’Neill, Jens Z. Pedersen, Oliviero Carugo, Giuseppe Rotilio, Andrea Battistoni, Kristina Djinović‐Carugo,
Tópico(s)Metal complexes synthesis and properties
ResumoThe sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-Å resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD. The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-Å resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD. Tuberculosis is the major cause of death in the world due to infection with a single microbial agent (1Zahrt T.C. Microbes Infect. 2003; 2: 159-167Crossref Scopus (71) Google Scholar). To ultimately combat this disease, concerted international efforts have been undertaken concerning the structural genomics of its etiological agent, Mycobacterium tuberculosis (2Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6445) Google Scholar). 1Available at www.doe-mbi.ucla.edu/TB/ and www.pasteur.fr/recherche/X-TB/. 1Available at www.doe-mbi.ucla.edu/TB/ and www.pasteur.fr/recherche/X-TB/.M. tuberculosis is a facultative intracellular pathogen capable of persisting in the highly oxidative environment of macrophages due to its resistance to the reactive oxygen and nitrogen species generated by phagocytic cells as part of their antimicrobial response (3Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Crossref PubMed Scopus (1143) Google Scholar). The resistance of M. tuberculosis to such reactive molecules is mediated by specific components of the cell wall, such as phenolic glycolipids and cyclopropanated mycolic acids, as well as by many mycobacterial antioxidant enzymes, including the two superoxide dismutases (SODs) 2The abbreviations used are: SOD, superoxide dismutase; MtSOD, M. tuberculosis superoxide dismutase; Cu,Zn-SOD, copper/zinc-containing superoxide dismutase; ApSOD, A. pleuropneumoniae superoxide dismutase; XlSOD, X. laevis superoxide dismutase; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. 2The abbreviations used are: SOD, superoxide dismutase; MtSOD, M. tuberculosis superoxide dismutase; Cu,Zn-SOD, copper/zinc-containing superoxide dismutase; ApSOD, A. pleuropneumoniae superoxide dismutase; XlSOD, X. laevis superoxide dismutase; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. encoded by the sodA and sodC genes.SODs are widely distributed enzymes that are classified according to the metal cofactors involved in the redox reaction, which catalyze the disproportionation of the superoxide radical ( O2−) into molecular oxygen (O2) and hydrogen peroxide (H2O2) (4Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2689) Google Scholar). As the investigation of the strategies used by M. tuberculosis to withstand the toxicity of reactive oxygen species may be considered of interest for the development of novel anti-tubercular strategies, several studies have been focused on the role and structure of mycobacterial SODs. M. tuberculosis sodA encodes an iron-containing SOD that is actively secreted to inhibit the host responses to mycobacterial infections (5Edwards K.M. Cynamon M.H. Voladri R.K. Hager C.C. DeStefano M.S. Tham K.T. Lakey D.L. Bochan M.R. Kernodle D.S. Am. J. Respir. Crit. Care Med. 2001; 164: 2213-2219Crossref PubMed Scopus (160) Google Scholar). The structure of this enzyme is very similar to that of other iron- or manganese-containing bacterial SODs (6Cooper J.B. McIntyre K. Badasso M.O. Wood S.P. Zhang Y. Garbe T.R. Young D. J. Mol. Biol. 1995; 246: 531-544Crossref PubMed Scopus (89) Google Scholar). On the other hand, sodC encodes a membrane-bound enzyme (7D'Orazio M. Folcarelli S. Mariani F. Colizzi V. Rotilio G. Battistoni A. Biochem. J. 2001; 359: 17-22Crossref PubMed Scopus (36) Google Scholar) that helps M. tuberculosis to survive in macrophages that generate a robust oxidative burst (8Piddington D.L. Fang F.C. Laessig T. Cooper A.M. Orme I.M. Buchmeier N.A. Infect. Immun. 2001; 69: 4980-4987Crossref PubMed Scopus (222) Google Scholar). This is in agreement with growing observations showing that sodC-null mutants of several pathogens are attenuated in animal models (9Battistoni A. Biochem. Soc. Trans. 2003; 31: 1326-1329Crossref PubMed Google Scholar). An additional reason to consider the sodC-encoded SOD (hereafter referred to as MtSOD) to be important for the protection of M. tuberculosis from reactive oxygen species generated by macrophages is that sodC is up-regulated upon entry into human macrophages, when superoxide production by NADPH oxidase is expected to be very high (7D'Orazio M. Folcarelli S. Mariani F. Colizzi V. Rotilio G. Battistoni A. Biochem. J. 2001; 359: 17-22Crossref PubMed Scopus (36) Google Scholar). In contrast, transcription of sodC is repressed during mycobacterial non-replicating persistence in mice, when phagocytic NADPH oxidase activity drops to very low levels (10Shi L. Jung Y.J. Tyagi S. Gennaro M.L. North R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 241-246Crossref PubMed Scopus (219) Google Scholar).Copper/zinc-containing SODs (Cu,Zn-SODs) are characterized by a conserved arrangement of the redox center, where Cu2+ is coordinated by four histidines, and the zinc ion is bound by three histidines and one aspartic acid. The two metal ions are simultaneously coordinated by a common histidine ligand (termed the "histidine bridge" or the "bridging imidazolate"), forming a structural motif found so far only in Cu,Zn-SODs (11Tainer J.A. Getzoff E.D. Beem K.M. Richardson J.S. Richardson D.C. J. Mol. Biol. 1982; 160: 287-303Crossref PubMed Scopus (895) Google Scholar). The copper ion is cyclically reduced and oxidized during successive encounters with the substrate (12McAdam M.E. Fielden E.M. Lavelle F. Calabrese L. Cocco D. Rotilio G. Biochem. J. 1977; 167: 271-274Crossref PubMed Scopus (71) Google Scholar), whereas the zinc ion has a very important structural role (13Forman H.S. Fridovich I. J. Biol. Chem. 1973; 248: 2645-2649Abstract Full Text PDF PubMed Google Scholar) and has also been shown to finely modulate the redox properties of the copper ion (14Estevez A.G. Crow J.P. Sampson J.B. Reiter C. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Crossref PubMed Scopus (509) Google Scholar). Although its primary sequence shows some unusual features, the sodC-encoded enzyme is classified as a Cu,Zn-SOD due to its sequence homology to other enzymes belonging to this family (2Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6445) Google Scholar, 15Bordo D. Matak D. Djinovic-Carugo K. Rosano C. Pesce A. Bolognesi M. Stroppolo M.E. Falconi M. Battistoni A. Desideri A. J. Mol. Biol. 1999; 285: 283-296Crossref PubMed Scopus (60) Google Scholar).Here, we present the crystal structure of the sodC-encoded enzyme from M. tuberculosis at 1.63-Å resolution, which was solved by the molecular replacement method using yeast Cu,Zn-SOD as a search model. Albeit this enzyme shows clear structural homology to the other enzymes of the Cu,Zn-SOD family, it was found to be fully functional despite the lack of the zinc ion in the active site and consequent structural rearrangements. The structure additionally presents a novel intersubunit interface distinct from those so far observed for dimer interactions in eukaryotic and prokaryotic Cu,Zn-SODs.EXPERIMENTAL PROCEDURESMtSOD Expression and Purification—Soluble recombinant MtSOD was purified from Escherichia coli 71/18 cells harboring plasmid pΔCysMycSOD (7D'Orazio M. Folcarelli S. Mariani F. Colizzi V. Rotilio G. Battistoni A. Biochem. J. 2001; 359: 17-22Crossref PubMed Scopus (36) Google Scholar). Cells were grown in LB broth supplemented with 125 μm CuSO4, and MtSOD expression was induced in the early logarithmic phase by the addition of 4 μm isopropyl-β-d-thiogalactopyranoside. Three hours after induction, cells were harvested by centrifugation, and the periplasmic proteins were extracted as described previously (16Battistoni A. Folcarelli S. Cervone L. Polizio F. Desideri A. Giartosio A. Rotilio G. J. Biol. Chem. 1998; 273: 5655-5661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Proteins were dialyzed against 20 mm Tris-HCl (pH 7.0), loaded onto a HiLoad 16/10 Q-Sepharose fast protein liquid chromatography column (Amersham Biosciences), and fractionated with a 0-150 mm NaCl linear gradient. Fractions containing MtSOD were concentrated and injected onto a HiLoad 16/60 Superdex 75 fast protein liquid chromatography gel filtration column (Amersham Biosciences) equilibrated with 20 mm Tris-HCl (pH 7.0) and 0.15 m NaCl. MtSOD eluted in a single peak with an apparent molecular mass close to 50 kDa. As a final step, the enzyme was further subjected to ion-exchange chromatography on a HiLoad 16/10 Q-Sepharose column (Amersham-Biotech) using a 0-0.2 m NaCl linear gradient in 20 mm Tris-HCl (pH 7.8). At this stage, the protein appeared to be >95% homogeneous, as judged by SDS-PAGE analysis. Protein concentration was evaluated by the method of Lowry et al. (17Lowry O.H. Rosebrough N.J. Farr L.A. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. To determine the metal content of MtSOD, the purified protein was extensively dialyzed against Chelex 100-treated 10 mm phosphate buffer (pH 7.0) and then analyzed by atomic absorption spectroscopy using a PerkinElmer Life Sciences AAnalyst 300 spectrometer equipped with a HGA-800 graphite furnace. The copper content was found to be between 0.6 and 0.8 eq/enzyme subunit depending on the preparations, whereas only a trace amount of zinc was found.Activity Assays—SOD activity assays as a function of pH and ionic strength were carried out by the pulse radiolysis method (18Fielden E.M. Roberts P.B. Bray R.C. Lowe D.J. Mautner G.N. Rotilio G. Calabrese L. Biochem. J. 1974; 139: 49-60Crossref PubMed Scopus (303) Google Scholar) as recently described (19Pesce A. Battistoni A. Stroppolo M.E. Polizio F. Nardini M. Kroll J.S. Langford P.R. O'Neill P. Sette M. Desideri A. Bolognesi M. J. Mol. Biol. 2000; 302: 465-478Crossref PubMed Scopus (43) Google Scholar).Spectroscopy—Electronic absorption studies were carried out using a Beckman Coulter DU 800 UV-visible spectrophotometer. EPR spectra were recorded at 298 K with a Bruker ESP300 spectrometer operating at 9.82 GHz using a high sensitivity TM110 cavity and flat glass capillaries. The protein copper concentration was 1 mm in 20 mm Tris-HCl. The pH titration was carried out by adding small amounts of 1 m NaOH; the pH was determined before and after each EPR measurement using a pH microelectrode.Demetallation/Remetallation of MtSOD—To remove metals from MtSOD, 10 mg/ml samples of the enzyme were initially dialyzed for 24 h at 4 °C against 50 mm sodium acetate buffer (pH 3.8) and 2 mm EDTA and then for 24 h against 50 mm sodium acetate (pH 3.8) and 0.1 m NaCl to remove EDTA. These samples were subsequently dialyzed twice against 50 mm sodium acetate buffer (pH 5.0) and finally against 50 mm sodium acetate buffer (pH 5.5). The complete absence of zinc and copper ions was verified by atomic absorption analysis. To investigate the presence of a functional zinc-binding site, CoCl2 or ZnCl2 was added to the copper-reconstituted enzyme, and metal uptake was checked by monitoring changes in the electronic and EPR spectra and by atomic absorption analyses.Crystallization—Crystallization setups utilizing the hanging drop vapor diffusion method produced well diffracting crystals in the form of plates in a couple of days. For all crystallization experiments, a single batch of MtSOD preparation with a concentration of 10 mg/ml was used. The optimal crystallization condition is 30% polyethylene glycol 4000, 200 mm ammonium sulfate, 20 mm ZnCl2, and 100 mm Tris (pH 8.2).Data Collection and Data Processing—Prior to data collection, the crystals were transferred to a cryoprotecting solution containing 20% glycerol and a slightly elevated polyethylene glycol 4000 concentration. The crystals were flash-frozen and kept under a 100 K dry nitrogen stream during data acquisition. The diffraction data were collected at the ID13 microfocus beamline of the European Synchrotron Radiation Facility, where the fine focus of the x-ray beam allows collection of data from small but perfect regions of slightly imperfect crystals. 180 rotation images with a 1° oscillation angle were collected in one pass without significant overloads in the low resolution regime. The data were integrated and scaled by XDS (20Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3213) Google Scholar) and were converted to CCP4 MTZ format. Statistics of data collection are shown in Table I.Table IData processing and refinement statisticsResolution range (outer shell) (Å)27.95 to 1.63 (1.80 to 1.63)Completeness (%)96.0 (87.9)〈I/σI〉13.5 (5.2)Rsym (%)aRsym=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl〈(hkl)〉, where Ii is the intensity of the ith measurement of reflection hkl and 〈I〉 is the average intensity of these i reflections.6.7 (27.3)No. reflections20248R-factor (%)bR−factor=∑hkl|Fo|−k|Fc|/∑hkl|Fo|, where k is a scaling factor. Rfree is calculated in the same way with 5% of the reflections that were omitted from refinement.15.0Rfree (%)bR−factor=∑hkl|Fo|−k|Fc|/∑hkl|Fo|, where k is a scaling factor. Rfree is calculated in the same way with 5% of the reflections that were omitted from refinement.19.0Correlation coefficient between Fo and Fc0.969Ramachandran plot Core (allowed) (%)89.5 (10.5)r.m.s.d.cRoot mean square deviation. from idealityBonds (Å)0.010Angles1.36°No. atoms (B-factor) (Å2)Non-H protein atoms1198 (15.03)Copper ions1 (20.75)Water272 (31.28)a Rsym=∑hkl∑i|Ii(hkl)−〈I(hkl)〉|/∑hkl〈(hkl)〉, where Ii is the intensity of the ith measurement of reflection hkl and 〈I〉 is the average intensity of these i reflections.b R−factor=∑hkl|Fo|−k|Fc|/∑hkl|Fo|, where k is a scaling factor. Rfree is calculated in the same way with 5% of the reflections that were omitted from refinement.c Root mean square deviation. Open table in a new tab Structure Solution and Refinement—The crystal structure of MtSOD was solved by molecular replacement utilizing the structure of a Cu,Zn-SOD from yeast (Protein Data Bank code 1JCV) as a search model. The first solution peak of the rotation function in MOLREP (21Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4118) Google Scholar) was identified as the correct solution with nearly 2-fold peak height compared with the rest. The molecular replacement solution was used as input to the ARP/wARP program suite (22Perrakis A. Morris R.J. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2562) Google Scholar) without any modification to the template sequence. In the first step, 25 cycles of the protocol "Improvement of Model by Atom Update and Refinement" were applied, followed by 50 cycles of refinement with automatic main chain tracing at the end of each 10 refinement cycles. This procedure resulted in a protein structure of 162 residues of two peptide chains. The correct side chains were unambiguously docked by the helper program GUISIDE. Nine additional residues were manually added to the structure. The refinement of the structure was carried out with REFMAC Version 5.1.24 (23Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13774) Google Scholar) as included in CCP4 Version 4.2.2 (24Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19702) Google Scholar). Water molecules were added to the structure by ARP/wARP (CCP4 version) after each of five REFMAC cycles. The refinement of the structure was completed when correct geometry and good correlation with diffraction data had been reached as judged from root mean square deviation from ideal geometry, R- and free R-factors, respectively. The structure was validated prior to submission to the Protein Data Bank using PROCHECK (25Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 265: 283-291Crossref Google Scholar) and SFCheck (26Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. Sect. D. 1999; 55: 191-205Crossref PubMed Scopus (857) Google Scholar). Details of refinement statistics are shown in Table I.Structure-based Alignment and Structural Comparisons—The structure-based alignment was generated with the program TOPP from the CCP4 program suite (27Lu G. Protein Data Bank Quarterly Newsletter. 1996; 78: 10-11Google Scholar) using an MtSOD monomer on which the structures of Actinobacillus pleuropneumoniae SOD (ApSOD) and Xenopus laevis SOD (XlSOD) monomers were superimposed. The program TOPP was set to the default parameters, counting a pair of residues as matching residues if there are at least three residues in a consecutive fragment of which C-α atoms are within 3.8 Å in the superimposed structures.Structural comparisons and classifications were performed with the STRUCLA metaserver (28Sasin J.M. Kurowski M.A. Bujnicki J.M. Bioinformatics. 2003; 19: 252-254Crossref PubMed Scopus (9) Google Scholar). A default distance cutoff of 3.5 Å was used to define atomic equivalencies after optimal superposition. Phylogenetic trees were built and analyzed with TreeView (29Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar).RESULTSThe sodC gene of M. tuberculosis encodes an SOD of 240 residues (Swiss-Prot accession number P96278), which is posttranslationally modified by cleaving a 32-amino acid long leader peptide from the N terminus. The mature protein is anchored to the bacterial membrane via a glyceride/fatty acid lipid attached to its N-terminal cysteine residue (7D'Orazio M. Folcarelli S. Mariani F. Colizzi V. Rotilio G. Battistoni A. Biochem. J. 2001; 359: 17-22Crossref PubMed Scopus (36) Google Scholar). To facilitate the biochemical characterization of the enzyme, the mature protein was expressed as a periplasmic soluble form in E. coli. The numbering of the sequence starts from the N-terminal cysteine of the mature protein as shown in Fig. 1, and this numbering scheme is used throughout.Crystallographic Analysis—The refined model of MtSOD reported here consists of one monomer containing a total of 170 residues, one copper ion, and 252 water molecules. No zinc ion is present in the crystal structure, although Zn2+ had been added to the crystallization mixture. The occupancy of the copper ion was set to 0.75 in agreement with σA-weighted (2Fo - Fc and Fo - Fc) difference density maps. The crystallographic R-factor and free R-factor for the refined model are 0.150 and 0.190 for the diffraction data in the 27.95- to 1.63-Å resolution range (Table I). The electron density is clearly defined for residues 38-208, whereas residues 1-37 are not visible in the electron density.Overall Structure and Comparison with Other Cu,Zn-SOD Structures—MtSOD adopts the eight-stranded antiparallel β-barrel with Greek key topology that is typical of both eukaryotic and prokaryotic Cu,Zn-SODs (30Bordo D. Pesce A. Bolognesi M. Stroppolo M.E. Falconi M. Desideri A. Messerschmidt A. Huber R. Poulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley & Sons Ltd., Chichester, United Kingdom2001: 1284-1300Google Scholar). Such a topological organization allows the closure of the β-barrel through two external crossing loops (Fig. 2A). Residue 38 is at the beginning of β-strand 1a of the classical Cu,Zn-SOD scaffold (Figs. 1 and 2A). The preceding 37 residues are probably not ordered due to the extreme flexibility of this N-terminal region, which is involved in the connection to the membrane (7D'Orazio M. Folcarelli S. Mariani F. Colizzi V. Rotilio G. Battistoni A. Biochem. J. 2001; 359: 17-22Crossref PubMed Scopus (36) Google Scholar). The structure-based alignment shown in Fig. 1 and superposition of MtSOD with ApSOD and XlSOD (Fig. 2B) show the special features of the sodC-encoded SOD from M. tuberculosis with respect to the prokaryotic and eukaryotic SOD enzymes.Fig. 2A, schematic view of the tertiary structure of MtSOD showing the characteristic structural elements of all Cu,Zn-SODs and the dimerization loop, a unique feature of MtSOD. Helices and β-strands are green, and loops are yellow. The figure was prepared by Molscript (56Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Rastrer3d (57Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar). B, superposition of MtSOD (green), ApSOD (blue), and XlSOD (red) made by TOPP. The superimposed proteins are oriented according to A. The figure was prepared by PyMOL (58DeLano W.L. PyMOL. DeLano Scientific, San Carlos, CA2002Google Scholar). C, phylogenetic tree of the SODs from prokaryotes, eukaryotes, and M. tuberculosis obtained by the consensus approach implemented in the STRUCLA metaserver. term, terminus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structural comparisons and classifications of MtSOD with representative structures of eukaryotic and prokaryotic Cu,Zn-SODs were performed with the STRUCLA metaserver (28Sasin J.M. Kurowski M.A. Bujnicki J.M. Bioinformatics. 2003; 19: 252-254Crossref PubMed Scopus (9) Google Scholar), which makes a consensus phylogenetic analysis (Fig. 2C) based on various measures of structural divergence between pairs of three-dimensional models, including PRIDE (31Carugo O. Pongor S. J. Mol. Biol. 2002; 315: 887-898Crossref PubMed Scopus (88) Google Scholar), rmsd_100 (32Carugo O. Pongor S. Protein Sci. 2001; 10: 1470-1473Crossref PubMed Scopus (233) Google Scholar), the classical root mean square deviation, the Johnson approach (33Johnson M.S. Stutclife M.J. Blundell T.L. J. Mol. Evol. 1990; 30: 43-59Crossref PubMed Scopus (92) Google Scholar) and the Grishin procedure (34Grishin N.V. J. Mol. Evol. 1997; 45: 359-369Crossref PubMed Scopus (35) Google Scholar). Two completely independent measurements of structural divergence (PRIDE and rmsd_100) are shown in the Tables II and III. rmsd_100 is the root mean square deviation that would have been observed after optimal superposition if the proteins being compared had 100 residues, and PRIDE is a probability value based on C-α-C-α distance comparison. The perfect matching between three-dimensional structures of two proteins is associated with values for rmsd_100 of 0.0 and for PRIDE of 1.00. Large rmsd_100 values indicate very divergent models as well as PRIDE values close to 0.0.Table IIAverage PRIDE values (standard deviations on the last digit in parentheses) obtained by comparing the SODs of prokaryotes, eukaryotes, and M. tuberculosisPRIDE valuesProkaryoticEukaryoticM. tuberculosisProkaryotic0.89 (4)0.77 (2)0.63 (6)Eukaryotic0.77 (2)0.96 (1)0.76 (2)M. tuberculosis0.63 (6)0.76 (2) Open table in a new tab Table IIIAverage rmsd_100 values (standard deviations on the last digit in parentheses) obtained by comparing the SODs of prokaryotes, eukaryotes, and M. tuberculosisrmsd_100 valuesProkaryoticEukaryoticM. tuberculosisProkaryotic0.27 (2)0.70 (1)0.60 (2)Eukaryotic0.70 (1)0.23 (1)0.63 (1)M. tuberculosis0.60 (2)0.63 (1) Open table in a new tab Active Site and Zinc Subloop—The active site of Cu,Zn-SODs is located at the bottom of an ∼7-Å long protein channel built by residues belonging to the S-S subloop and residues belonging to the electrostatic loop. In MtSOD, the copper ion is coordinated by His84, His86, His163, and a water molecule. The distances of the copper ion from these residues are 2.07 Å (Cu-His84 N-δ1), 2.05 (Cu-His86 N-ϵ2), 2.04 Å (Cu-His163 N-ϵ2), and 1.93 Å (Cu-H2O), whereas the fourth putative ligand (His112 N-ϵ2), which corresponds to the bridging ligand present in all the other Cu,Zn-SODs, is situated 3.29 Å from the copper ion (Fig. 1 and 3A).Fig. 3A, active center of MtSOD showing the copper ion and its coordinating residues as well as electron density of a 2Fo - Fc map contoured at the 1.4σ level. Besides the residues involved in coordination (connected to copper with dashed lines), the universally conserved Arg199 (Arg141 in bovine SOD), the "bridging" His112 (His61 in bovine SOD), and a water molecule with connectivity to the copper ligand (W448) are shown. The figure was prepared by PyMOL (58DeLano W.L. PyMOL. DeLano Scientific, San Carlos, CA2002Google Scholar). B, superposition of MtSOD (red) and ApSOD (blue) demonstrating the differences between the zinc subloops of mycobacterial and classical bacterial Cu,Zn-SODs. Conserved residues in Cu,Zn-SODs (bovine numbering) are labeled in black; residues mutated or unique in MtSOD are labeled in red. The zinc subloop of MtSOD is significantly shorter and lacks some of the residues indispensable for zinc binding such as His69 and His78 (bovine numbering). His69 is missing due to deletion of part of the zinc subloop in MtSOD, whereas His78 is mutated to Ala123 in all mycobacterial SODs. The positions of the two histidine residues (His114 and His118) in the short zinc loop of MtSOD exclude zinc binding. The figure was prepared by Molscript (56Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Rastrer3d (57Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar). term, terminus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutations and deletions of conserved residues involved in zinc coordination were analyzed by superposition of zinc subloops (Fig. 3B) in copper/zinc-containing ApSOD and MtSOD. Two conserved zinc-binding histidine residues are missing in MtSOD: position 123 hosts an Ala, whereas His69 (bovine numbering) is lost because of a seven-residue deletion in the zinc subloop. This deletion brings about structural rearrangements that involve also the second coordination sphere of the catalytic metal.Dimer Architecture—All eukaryotic Cu,Zn-SODs have a tight and stable dimeric structure based on a conserved subunit interface that is formed by 19 residues coming from β-strands 1a, 2b, and 8h, from the S-S subloop, and from the Greek key subloop, and by the C-terminal region (Fig. 2A). The quaternary structure assembly in prokaryotic Cu,Zn-SODs is entirely different and is based on 18 residues provided by β-strands 4f and 5e and by loops
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