Structure-Function Relationship of the Chloroplastic Glutaredoxin S12 with an Atypical WCSYS Active Site
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m807998200
ISSN1083-351X
AutoresJérémy Couturier, Cha San Koh, Mirko Zaffagnini, Alison M. Winger, José M. Gualberto, Catherine Corbier, Paulette Decottignies, Jean‐Pierre Jacquot, Stéphane D. Lemaire, Claude Didierjean, Nicolas Rouhier,
Tópico(s)Trace Elements in Health
ResumoGlutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28WCSYS32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation. Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28WCSYS32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation. Glutaredoxins (Grxs) 5The abbreviations used are: Grx, glutaredoxin; βMSH, β-mercaptoethanol; DHA, dehydroascorbate; DTTred, reduced dithiothreitol; DTTox, oxidized dithiothreitol; GR, glutathione reductase; HED, hydroxyethyldisulfide; Trx, thioredoxin; WT, wild type; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; PDT-bimane, (2-pyridyl)dithiobimane; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; r.m.s., root mean square; ISC, iron-sulfur cluster. are GSH- or thioredoxin reductase-dependent oxidoreductases involved in the maintenance of cellular redox homeostasis. When Grxs are recycled by GSH, the GSSG formed is in turn reduced by NADPH and glutathione reductase (GR), forming the GSH/Grx reducing system. The first Grxs characterized usually contained the active site motif Cys-Pro-Tyr-Cys, the second active site cysteine being generally not essential for Grx activity (1Bushweller J.H. Aslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar, 2Rouhier N. Gelhaye E. Jacquot J.P. FEBS Lett. 2002; 511: 145-149Crossref PubMed Scopus (63) Google Scholar). It has been shown, however, to be required for a few reactions, such as the reduction of some low molecular weight disulfides or of disulfide bonds in E. coli ribonucleotide reductase and phosphoadenylyl-sulfate reductase (1Bushweller J.H. Aslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar, 3Lillig C.H. Prior A. Schwenn J.D. Aslund F. Ritz D. Vlamis-Gardikas A. Holmgren A. J. Biol. Chem. 1999; 74: 7695-7698Abstract Full Text Full Text PDF Scopus (121) Google Scholar, 4Johansson C. Lillig C.H. Holmgren A. J. Biol. Chem. 2004; 279: 7537-7543Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). In most cases, Grxs rather reduce specifically protein-glutathione adducts via two distinct mechanisms. The monothiol mechanism requires only the more N terminus active site cysteine together with two glutathione molecules, and the dithiol mechanism requires either the two active site cysteines or the N terminus active site cysteine and a conserved extra active site C terminus cysteine. Both types of disulfides formed on Grx are reduced in vitro by GSH or thioredoxin reductases (1Bushweller J.H. Aslund F. Wuthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (204) Google Scholar, 4Johansson C. Lillig C.H. Holmgren A. J. Biol. Chem. 2004; 279: 7537-7543Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 5Tamarit J. Bellí G. Cabiscol E. Herrero E. Ros J. J. Biol. Chem. 2003; 278: 25745-25751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In comparison, thioredoxins (Trxs) efficiently reduce protein disulfides but have low or no activity with mixed disulfides (7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Jung C.H. Thomas J.A. Arch. Biochem. Biophys. 1996; 335: 61-72Crossref PubMed Scopus (161) Google Scholar). Sharing high structural and functional homologies with Trx, Grxs display a common Trx fold (9Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). The CXXC motif in Trx and Grx is located in a partially exposed surface loop downstream of a β-strand and at the N terminus of an α-helix (9Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). Interestingly, some Trxs and Grxs only contain the N terminus cysteine of the CXXC motif, the second cysteine being very often replaced by a serine. In plants, this variation of active site sequence exists in approximately half of the 30 Grxs existing (10Rouhier N. Gelhaye E. Jacquot J.-P. Cell. Mol. Life Sci. 2004; 61: 1266-1277Crossref PubMed Scopus (174) Google Scholar). Grxs were initially categorized, based on the active site sequence, into two groups, a dithiol (CPY/FC motif) and a monothiol (CGFS motif) subgroup (11Rodríguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (265) Google Scholar). Nevertheless, the increasing number of Grxs with primary structures that deviate from these standard motifs has led to the proposal of a more appropriate and thorough classification of plant Grxs (10Rouhier N. Gelhaye E. Jacquot J.-P. Cell. Mol. Life Sci. 2004; 61: 1266-1277Crossref PubMed Scopus (174) Google Scholar, 12Lemaire S.D. Photosynth. Res. 2004; 79: 305-318Crossref PubMed Scopus (122) Google Scholar, 13Rouhier N. Couturier J. Jacquot J.-P. J. Exp. Bot. 2006; 57: 1685-1696Crossref PubMed Scopus (134) Google Scholar). Three subclasses have been defined based on their active site structures. Subclass I includes proteins with CXX(C/S) active sites other than CGFS, subclass II contains exclusively Grxs with a CGFS motif, and subclass III, which is specific to land plants, corresponds to Grxs with a peculiar CCXX active site. Many structures of dithiol Grxs from human (Protein Data Bank codes 1JHB, 2CQ9, 2FLS, 2HT9, 1B4Q), pig (Protein Data Bank code 1KTE), mouse (PDB codes 1T1V, 1WJK), plant (Protein Data Bank codes 1Z7P, 1Z7R, 2E7P), yeast (Protein Data Bank code 2JAC), bacteria (Protein Data Bank codes 1EGR, 1EGO, 1FOV, 1GRX, 1G7O, 1H75, 2AYT, 1J08, 1R7H, 2YWM), virus (Protein Data Bank codes 2HZE, 2HZF), or T4 bacteriophage (Protein Data Bank codes 1DE1, 1DE2, 1AAZ, 1ABA, 1QFN, 3GRX) have been solved by NMR spectroscopy or x-ray crystallography both in the oxidized and reduced forms. Some of these Grx structures contain either a low molecular weight substrate or an interacting peptide partner. Only one NMR structure (Escherichia coli Grx4, PDB code 1YKA) of monothiol Grx is available despite the abundance of genes reported. In this study, we have investigated the structure-function relationship of a monothiol Grx isoform (GrxS12) found in Populus tremula × tremuloides, which possesses an unusual 28WCSYS32 active site sequence, unique to plants. Phylogenetic analyses indicate that GrxS12 belongs to Grx subclass I, along with classical dithiol Grxs. In addition to the active site cysteine, there is an additional C terminus cysteine in position 87, which is present in many dithiol or monothiol Grxs. Its role remains obscure, although it has been demonstrated that it can serve as a resolving cysteine of the glutathionylated catalytic cysteine in a few organisms (5Tamarit J. Bellí G. Cabiscol E. Herrero E. Ros J. J. Biol. Chem. 2003; 278: 25745-25751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Biochemical and enzymatic studies of mutated proteins together with the resolution of liganded GrxS12 structures allowed us (i) to identify the GSH binding site of this enzyme, (ii) to investigate the role of the C terminus cysteine in the catalytic mechanism of GrxS12, and (iii) to understand why an iron-sulfur cluster is not present in GrxS12 despite its apparently favorable CSYS motif. Materials-NAP-5 columns were purchased from GE Healthcare. Hydroxyethyldisulfide (HED) and 5,5′-dithiobis-2-nitrobenzoic acid were from Aldrich and Pierce, respectively. All other reagents were from Sigma. Cloning and Construction of GrxS12 Mutants by Site-directed Mutagenesis-The open reading frame sequence encoding poplar GrxS12 was amplified from a P. tremula × tremuloides leaf cDNA library using GrxS12 forward and reverse primers (supplemental Table 1) and cloned into the NcoI and BamHI restriction sites (underlined in the primers) of pET3d (Novagen). The sequence amplified encodes a protein deprived of the first 74 amino acids corresponding to the putative targeting sequence and in which a methionine and an alanine have been added during cloning. The protein starts thus with the N terminus sequence 1MASFGSRL8 and ends with 98AKKSQG113 at the C terminus (see supplemental Fig. 1). Using two complementary mutagenic primers, the two cysteines of GrxS12 were individually substituted into serines, the Trp in position 28 was mutated into Tyr, and the active site was also entirely modified from WCSYS into YCGYC. The primers are listed in supplemental Table 1. The mutated proteins are called GrxS12 W28Y, C29S, and C87S and GrxS12 YCGYC. Expression and Purification of the Recombinant Proteins-For protein production, the E. coli BL21(DE3) strain, containing the pSBET plasmid, was co-transformed with the different recombinant plasmids (14Schenk P.M. Baumann S. Mattes R. Steinbiss H.H. BioTechniques. 1995; 19: 196-200PubMed Google Scholar). Cultures were successively amplified up to 2.4 l in LB medium supplemented with ampicillin and kanamycin at 37 °C. Protein expression was induced at exponential phase by adding 100 μm isopropyl β-d-thiogalactopyranoside for 4 h at 37 °C. The cultures were then centrifuged for 15 min at 4400 × g. The pellets were resuspended in 30 ml of TE NaCl (30 mm Tris-HCl, pH 8.0, 1 mm EDTA, 200 mm NaCl) buffer, and the suspension was conserved at -20 °C. Cell lysis was performed by sonication (3 × 1 min with intervals of 1 min), and the soluble and insoluble fractions were separated by centrifugation for 30 min at 27,000 × g. The soluble part was then fractionated with ammonium sulfate in two steps, and the protein fraction precipitating between 40 and 80% of the saturation contained the recombinant protein, as estimated by 15% SDS-PAGE. The protein was purified by size exclusion chromatography after loading on an ACA44 (5 × 75-cm) column equilibrated in TE NaCl buffer. The fractions containing the protein were pooled, dialyzed by ultrafiltration to remove NaCl, and loaded onto a DEAE-cellulose column (Sigma) in TE (30 mm Tris-HCl, pH 8.0, 1 mm EDTA) buffer. All of the proteins (GrxS12 WT, W28Y, C29S, C87S, and YCGYC) passed through the DEAE column and were subsequently loaded onto a carboxymethylcellulose column (Sigma) in TE buffer. The proteins were eluted using a 0–0.4 m NaCl gradient. Finally, the fractions of interest were pooled, dialyzed, concentrated by ultrafiltration under nitrogen pressure (YM10 membrane; Amicon), and stored in TE buffer at -20 °C. Purity was checked by SDS-PAGE. Protein concentrations were determined spectrophotometrically using a molar extinction coefficient at 280 nm of 9970 m-1 cm-1 for the GrxS12 WT, C29S, and C87S and 5960 m-1 cm-1 for GrxS12 W28Y and GrxS12 YCGYC. In Vivo Subcellular Localization-A fragment of 285 nucleotides coding for the 95 first amino acids of GrxS12 was cloned in the 5′-part of the GFP coding sequence under the control of a double 35S promoter into the plasmid pCK-GFP3 using GrxS12 pCK forward and reverse primers (supplemental Table 1). Nicotiana benthamiana epidermal leaf cells were transfected by bombardment of the abaxial side of young leaves with tungsten particles coated with plasmid DNA. Images were obtained 18 h later with a Zeiss LSM510 confocal microscope. Stomata cells were preferentially imaged, because of their small size and because typically only one of the two guard cells is transfected and expresses the GFP construction, whereas the untransfected cell serves the role of internal negative control. Chloroplasts were visualized by the natural fluorescence of chlorophyll. Reduction and Oxidation of Wild-type and Mutated GrxS12-The proteins (50–100 μm) were reduced by 10 mm DTT for 1 h at 25 °C, followed by desalting on NAP-5 column pre-equilibrated with 30 mm Tris-HCl, pH 7.9. Oxidized Grxs were prepared by incubation of prereduced with 10 mm oxidized DTT or 1–5 mm GSSG for 1–2 h at 25 °C. GSSG-oxidized GrxS12 (WT or mutants) were desalted as above and treated by 10 mm reduced DTT or by 2 mm GSH in the presence of 6 μg/ml yeast glutathione reductase and 0.5 mm NADPH. Mass Spectrometry Analysis-Reduced and oxidized GrxS12 WT and C87S and trypsin-cleaved proteins were analyzed by MALDI-TOF MS as described in Ref. 15Augusto L. Decottignies P. Synguelakis M. Nicaise M. Le Maréchal P. Chaby R. Biochemistry. 2003; 42: 3929-3938Crossref PubMed Scopus (51) Google Scholar. Fluorescence Properties of Wild-type and Mutated GrxS12-The fluorescence characteristics of GrxS12 and GrxS12 C87S in the reduced and oxidized forms were recorded with a spectrofluorometer (Cary Eclipse; VARIAN) in TE buffer with a 10 μm concentration of each protein. Determination of Free Thiol Groups-The number of free thiol groups in untreated, reduced, or oxidized proteins was determined spectrophotometrically with 5,5′-dithiobis-2-nitrobenzoic acid, as described in Ref. 7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar. pKa Determination of GrxS12 Sulfhydryls with (2-pyridyl)dithiobimane (PDT-bimane)-The reaction of PDT-bimane with cysteine forms pyridine-2-thione, which has a maximum absorption wavelength of 343 nm (16Mansoor S.E. Farrens D.L. Biochemistry. 2004; 43: 9426-9438Crossref PubMed Scopus (50) Google Scholar). The stock solution of PDT-bimane was made in DMSO, and the concentration was determined using the absorbance extinction coefficient at 380 nm, ∈380 = 5000 m-1 cm-1 in ethanol. Reactions were started by the addition of PDT-bimane to a final concentration of 25 μm into a cuvette containing 10 μm reduced proteins in 500 μl of sodium citrate or phosphate buffer ranging from pH 3.0 to 8.0 and rapidly mixed, and the absorbance at 343 nm was recorded over 120 min with a Varian Cary 50 spectrophotometer. Absorbance data were fitted directly to the Michaelis-Menten equation with the GraphPad Prism 5 program and the t½ (the time to reach half-maximal reactivity as monitored by half-maximal release of pyridyl-2-thione) at each pH was determined. Those values were plotted against pH using sigmoidal curve fit and GraphPad Prism 5 (GraphPad software). Activity Measurements-The activity measurements of WT or mutant GrxS12 in the HED assay or for reduction of DHA or glutathionylated GAPDH were performed as described in Zaffagnini et al. (7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Crystallization, Data Collection, Structure Determination, and Crystallographic Refinement-The complex of poplar GrxS12 with glutathione (GrxS12·GSH) was directly obtained using the purified recombinant protein. The complex of GrxS12 with both GSH and β-mercaptoethanol (βMSH) (GrxS12·GSH·βMSH) was prepared after purification steps by the addition of 2 mm GSH and 10 mm HED to the purified recombinant protein for 2 h at room temperature prior to extensive dialyses against TE buffer to remove unbound GSH, HED, or βMSH. Crystals were grown at 20 °C by the microbatch under oil (paraffin) method. Optimal crystallization conditions were screened based on the sparse matrix crystallization approach. The protein at an initial concentration of 10–15 mg ml-1 in TE buffer was mixed with similar precipitant solutions (for each protein) in a 1:1 ratio. The GrxS12·GSH crystals were obtained by using 0.1 m Na-HEPES (pH 7.5) and 20% polyethylene glycol 8000 solution (JBS 5-B4), whereas GrxS12·GSH·βMSH crystallized in 0.1 m Na-HEPES (pH 7.5) and 25% polyethylene glycol 1000 solution (JBS 1-C3). Grx crystals grew rapidly within 2 days. X-ray diffraction data were collected from single crystals flash-cooled in a nitrogen stream at 100 K. Data of both complexes, GrxS12·GSH and GrxS12·GSH·βMSH, were collected on a MAR165 CCD detector at beamlines X11 and X13 (DESY/EMBL, Hamburg, Germany), respectively. All crystallographic data were indexed, processed, and scaled with HKL-2000 (17Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). Data collection and refinement statistics are summarized in Table 1. The crystal structure of GrxS12·GSH was determined by molecular replacement with MOLREP (18Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 19Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4174) Google Scholar), using human Grx2 (Protein Data Bank code 2FLS) as a model (20Johansson C. Kavanagh K.L. Gileadi O. Opperman U. J. Biol. Chem. 2007; 282: 3077-3082Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The resulting solution coordinates were used for automatic model building using the program ARP/wARP (21Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). After 75 cycles of autobuilding, 99% of the model (786 atoms were refined) was built automatically. Hence, the initial model of GrxS12·GSH comprises 100 residues with a connectivity index of 0.96 and R factor of 19.5%. This structure was then refined using REFMAC version 5.4 (18Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 22Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13911) Google Scholar) interspersed with manual inspection using COOT (23Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23605) Google Scholar). A GSH molecule was added almost toward the end of the refinement of the GrxS12·GSH model. The coordinates of GrxS12·GSH were then used to solve the structure of GrxS12·GSH·βMSH, also by molecular replacement. The structure refinement of the latter was done as for the template. Positions of water molecules were identified with ARP/wARP and were checked manually. The validation of both crystal structures was performed with PROCHECK (24Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All figures were prepared with PyMOL (25Delano W.L. PyMol. DeLano Scientific, Palo Alto, CA2002Google Scholar). Structure superimpositions were performed using the LSQMAN program from the DEJAVU package (26Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D. 1997; 53: 179-185Crossref PubMed Scopus (96) Google Scholar) or Lsqkab (superpose) program of the CCP4 package.TABLE 1Data collection and refinement statistics for GrxS12·GSH and GrxS12·GSH·βMSH crystalsData setGrxS12·GSHGrxS12·GSH·βMSHData collection and processing statisticsData collection siteX11 DESY/EMBL-HamburgX13 DESY/EMBL-HamburgWavelength (Å)0.81500.8063Space groupP212121P212121Unit cell dimensions (Å) (a, b, c)39.03, 47.27, 55.6238.83, 46.82, 55.36Asymmetric unit1 subunit1 subunitResolution range (Å)aRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively50.00–1.70 (1.73–1.70)50.00–1.80 (1.86–1.80)RedundancyaRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively4.49 (4.16)6.69 (6.85)Completeness (%)aRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively99.7 (95.0)99.6 (100.0)I/σIaRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively13.37 (2.26)23.26 (16.95)RmergeaRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively,bThe Rfree value was calculated from 5% of all data that were not used in the refinement0.045 (0.239)0.048 (0.107)Refinement statisticsResolution range (Å)36.0–1.7030.0–1.80Reflections used11,2349301RcrystcRcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively (Rfree)dRfree is as for Rcryst but calculated for a test set comprising reflections not used in the refinement (5%)19.28 (23.95)16.78 (21.45)Protein/waters/GSH/HED106 residues/186/1/0106 residues/186/1/0Mean B factor (Å2)Main chain16.5312.04Side chain23.3314.79Water32.5727.88Ligand15.3117.00All20.5216.24r.m.s. deviation from ideal geometryBond lengths (Å)0.0120.011Bond angles (degrees)1.41.4Dihedral angles (degrees)23.523.7Improper angles (degrees)1.611.75Ramachandran plotResidues in most favored regions (%)96.795.6Residues in additionally allowed regions (%)3.34.4Residues in generously allowed regions (%)0.00.0a Rcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelyb The Rfree value was calculated from 5% of all data that were not used in the refinementc Rcryst = Σ|Fo – Fc|/ΣFo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelyd Rfree is as for Rcryst but calculated for a test set comprising reflections not used in the refinement (5%) Open table in a new tab Subcellular Localization of GrxS12-The genome analysis of Populus trichocarpa suggests that at least four Grxs should be located in the chloroplast. Two Grxs of subgroup II, possessing a CGFS active site and called GrxS14 and GrxS16, have been experimentally shown to be located in plastids and would be involved in iron-sulfur biogenesis, since they incorporate an iron-sulfur cluster that can be transferred very quickly to apoferredoxin (27Bandyopadhyay S. Gama F. Molina-Navarro M.M. Gualberto J.M. Claxton R. Naik S.G. Huynh B.H. Herrero E. Jacquot J.P. Johnson M.K. Rouhier N. EMBO J. 2008; 27: 1122-1133Crossref PubMed Scopus (218) Google Scholar). The GrxS12 from P. tremula × tremuloides is a protein of 185 amino acids with a predicted chloroplastic N terminus-targeting sequence. In order to experimentally confirm its localization, the sequence coding the first 95 amino acids, including thus the putative targeting sequence and the first α-helix and β-strand, was fused to the GFP coding sequence and used to bombard tobacco leaf cells. As shown in Fig. 1, the fluorescence associated with this construction, transfected into one of the two guard cells of a stomate, strictly co-localizes with the autofluorescence of chlorophyll, indicating that the protein is indeed chloroplastic. Determination of GrxS12 Redox States by Fluorescence, Thiol Titration, and Mass Spectrometry-The recombinant protein produced in E. coli is a protein containing 113 residues devoid of the first 74 N terminus residues (which represent the transit peptide) and in which a methionine and an alanine have been added in the new N terminus end. The predicted molecular mass and pI are 12,360 Da and 8.56, respectively. A MALDI-TOF analysis of the purified GrxS12 WT revealed two protein peaks with molecular masses of 12,229.1 and 12,535.7 Da (data not shown). The first one is consistent with a protein where the methionine is cleaved, which is not surprising, since the second residue is an alanine, and the second peak is consistent with a glutathionylated protein also deprived of the methionine (306.6 Da mass increase compared with peak 1). There are two conserved cysteine residues in GrxS12, the active site cysteine in position 29 and a C terminus cysteine in position 87. In some CGFS Grxs, the latter cysteine can form an intramolecular disulfide with the catalytic cysteine (5Tamarit J. Bellí G. Cabiscol E. Herrero E. Ros J. J. Biol. Chem. 2003; 278: 25745-25751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 6Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 7Zaffagnini M. Michelet L. Massot V. Trost P. Lemaire S.D. J. Biol. Chem. 2008; 283: 8868-8876Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In order to check the possibility that Cys87 acts as a recycling cysteine in GrxS12, we measured the number of free thiol groups under reducing and oxidizing conditions in GrxS12 WT and C87S (Table 2). After purification and in the absence of any reducing or oxidizing treatment (native form), GrxS12 WT possesses about one free thiol group, whereas there is no free thiol in GrxS12 C87S. A reduction by DTTred led to the expected number of free thiols, two for the WT and one for the C87S mutant. After a prereduction step of WT GrxS12, a subsequent oxidation by DTTox over a long period (2 h) did not change these values, indicating that no intra- or intermolecular disulfide bridges can be formed. Such disulfides have not been observed on nonreducing SDS-polyacrylamide gels either (data not shown). On the contrary, subsequent oxidation using 1 mm GSSG gave the same values as those found originally in the untreated proteins, one free thiol group for GrxS12 WT but no free thiol in GrxS12 C87S. Together, these results suggest the presence of a glutathione adduct on the catalytic cysteine (Cys29), whereas the second cysteine (Cys87) is not modified. In order to confirm these results, we analyzed by MALDI-TOF mass spectrometry prereduced GrxS12 before or after glutathionylation treatments in the presence of GSSG or GSH and H2O2 (Fig. 2). After treatment, the mass of the protein increased by ∼305 Da, a feature consistent with the formation of one glutathione adduct. This increase was reversed by treatment with either DTTred or GSH/GR/NADPH. A similar behavior was observed for WT or C87S GrxS12, indicating that Cys29 is indeed the residue modified by glutathionylation. This was further confirmed by tryptic digestion and peptide mass fingerprinting of reduced or glutathionylated GrxS12. Indeed, the peptide (Thr27–Lys36) containing Cys29 is shifted by 305 Da in the glutathionylated protein. All of these results indicate that Cys29 is undergoing glutathionylation, whereas Cys87 is not glutathionylated in any of the conditions tested.TABLE 2Number of free thiols in WT and C87S GrxS12 under various redox conditions The untreated column is indicative of the protein thiol content measured after purification. GrxS12 was reduced by DTTred and subsequently oxidized by DTTox or GSSG. After each treatment (DTTred, DTTox, and GSSG), samples were desalted. The thiol content per protein was quantified by 5,5
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