research papers
Atomic resolution Plasmodium falciparum and comparison with other glutaredoxins
of glutaredoxin 1 fromaStructural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi 110 067, India, bDepartment of Biochemistry, North-Eastern Hill University, Shillong 792 022, India, cBiochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, 35392 Giessen, Germany, dEuropean Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble, France, and eUnit for Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 France
*Correspondence e-mail: amit.icgeb@gmail.com
Glutaredoxins (Grxs) are redox proteins that use glutathione (γGlu-Cys-Gly; GSH) as a cofactor. Plasmodium falciparum has one classic dithiol (CXXC) glutaredoxin (glutaredoxin 1; PfGrx1) and three monothiol (CXXS) Grx-like proteins (GLPs), which have five residue insertions prior to the active-site Cys. Here, the of PfGrx1 has been determined by the sulfur single-wavelength anomalous diffraction (S-SAD) method utilizing intrinsic protein and solvent S atoms. Several residues were modelled with alternate conformations, and an alternate position was refined for the active-site Cys29 owing to radiation damage. The GSH-binding site is occupied by water polygons and buffer molecules. Structural comparison of PfGrx1 with other Grxs and Grx-like proteins revealed that the GSH-binding motifs (CXXC/CXXS, TVP, CDD, Lys26 and Gln/Arg63) are structurally conserved. Both the monothiol and dithiol Grxs possess three conserved water molecules; two of these were located in the GSH-binding site. PfGrx1 has several polar and charged amino-acid substitutions that provide structurally important additional hydrogen bonds and salt bridges missing in other Grxs.
Keywords: glutaredoxin; glutathione; dithiol; S-SAD.
3D view: 4hjm,4kje,4kjf
PDB references: glutaredoxin 1, 4hjm; 4kje; 4kjf
1. Introduction
The intracellular redox milieu influences cellular functions, including those of gene expression, DNA synthesis, apoptosis and cellular signalling. Glutaredoxins (Grxs) are a family of small thiol–disulfide ; Holmgren et al., 2005; Lillig et al., 2008) and utilize electrons provided by the tripeptide glutathione (γGlu-Cys-Gly; GSH) to catalyze thiol–disulfide exchange reactions. Structurally, all Grxs share a common topological fold, the thioredoxin fold, which consists of four-stranded β-sheets flanked by three to five α-helices. Grxs were discovered as GSH-dependent hydrogen donors for ribonucleotide reductase in an Escherichia coli mutant lacking Trxs (Holmgren, 1976). Since then, Grxs have been assigned several other functions such as the reduction of methionine and sulfates (Gonzalez Porqué et al., 1970; Tsang, 1981). Grxs act as general thiol–disulfide and help to protect cells against oxidative stress by detoxifying oxidizing agents (Holmgren, 1979, 2000; Axelsson & Mannervik, 1980). Grxs are also involved in regulating transcription-factor binding activity (Matthews et al., 1992; Pineda-Molina et al., 2001), apoptosis (Chrestensen et al., 2000), redox-signal transduction and protein translocation (Shelton et al., 2005). Grxs catalyze important steps in oxidative protein folding by making protein–protein interactions and conducting covalent catalysis to act as chaperones and isomerases of disulfides (Berndt et al., 2008). Several Grxs bind to Fe–S clusters both in vitro and in vivo, thereby helping in intracellular iron sensing, enzyme catalysis and regulation (Lillig et al., 2005; Feng et al., 2006; Lill et al., 2006; Iwema et al., 2009; Hoff et al., 2009; Mühlenhoff et al., 2010). The functionally and mechanistically heterogeneous class of Grxs can be subdivided into monothiol (CXXS) and dithiol (CXXC) Grxs depending on the number of cysteine residues present in the active site. In parallel to this, two distinct mechanisms have been proposed to explain Grx activity based on one or two active-site cysteines. In the monothiol mechanism, only the N-terminal cysteine is required for reduction of Grx–GSH mixed disulfides, while in the dithiol mechanism Grxs can reduce both low-molecular-weight ligands and protein disulfides using both active-site cysteines (Fernandes & Holmgren, 2004; Gallogly et al., 2009). Both pathways use GSH as a reductant and concomitantly produce GSSG, which is in turn reduced back to GSH via glutathione reductase (GR) and NADPH.
that are ubiquitous and conserved in evolution. They are involved in the maintenance of thiol–disulfide redox equilibrium within cells (Fernandes & Holmgren, 2004Besides having three monothiol Grx-like proteins (GLPs), Plasmodium falciparum harbours a single gene encoding a typical dithiol Grx (PfGrx1), which is cytoplasmic (Rahlfs et al., 2001; Kehr et al., 2010). PfGrx1 displays glutathione:HEDS activity and can reduce ribonucleotide reductase and the thioredoxin-like protein plasmoredoxin in vitro (Rahlfs et al., 2001). PfGrx1 is highly thermostable and resistant to denaturants and pH changes (Tripathi et al., 2010). Several studies have identified interactions between Grxs and their partner proteins in plants, eukaryotic parasites and other organisms (Motohashi et al., 2001; Lindahl & Florencio, 2003; Balmer et al., 2003; Lemaire et al., 2004; Wong et al., 2004; Rouhier et al., 2005; Sturm et al., 2009). These interacting proteins are involved in many processes, including oxidative stress response (peroxiredoxins, ascorbate peroxidase and catalase), carbon/nitrogen/sulfur metabolism (S-adenosylmethionine synthetase, S-adenosyl-L-homocysteine hydrolase and phosphoglycerate kinase), protein biosynthesis (elongation factors) and protein folding (heat-shock proteins and protein disulfide isomerase). Recently, it has been shown that the parasite P. falciparum antioxidant protein (PfAOP; 1-Cys peroxiredoxin) requires PfGrx1 and glutathione as substrates for the reduction of (Djuika et al., 2013). In this work, we provide atomic resolution structural information on PfGrx1. The latter has several additional hydrogen bonds and salt bridges owing to polar and charged amino-acid substitutions when compared with other Grxs. PfGrx1 has a unique hydrophobic pocket filled with the crystallization solvent molecule MPD. We also show that the disulfide bond between Cys29 and Cys32 is broken up on exposure to synchrotron radiation. Finally, our analysis of residue conservation and surface electrostatic potential distributions in Grxs provides insights into the intriguing structural diversity of Grxs.
2. Materials and methods
2.1. Expression and purification of the recombinant protein
Recombinant PfGrx1 was produced as described by Rahlfs et al. (2001). Escherichia coli M15 cells were transformed with the respective pQE30grx plasmid. 5 ml of LB medium was inoculated with a single colony and used as a starter culture for a 500 ml culture. The cells were grown at 310 K in LB medium containing ampicillin (100 µg ml−1) and kanamycin (50 µg ml−1) to an A600 of 0.6; subsequently, protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were grown for an additional 4 h, harvested, and either directly used for protein purification or frozen at 253 K. For purification, bacterial cells were disintegrated via sonication in the presence of a protease-inhibitor cocktail. The protein was purified using Ni2+–NTA beads (Qiagen) and gel-filtration Fractions containing the protein were analyzed by SDS–PAGE (Supplementary Fig. S11). The purified protein was finally concentrated and the buffer was exchanged to 50 mM HEPES, 50 mM KCl, 1 mM EDTA pH 8.0.
2.2. Crystallization, sulfur-SAD and atomic resolution data collection
Crystallization experiments were performed using the hanging-drop vapour-diffusion method at 293 K. Initial crystallization screening was carried out in a Costar 96-well tissue-culture plate (Corning, Lowell, Massachusetts, USA) with ClearSeal Film (Hampton Research, USA) using the Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2 (Hampton Research, USA) and Morpheus (Molecular Dimensions, UK) screens. Three different drop ratios were aliquoted using a Mosquito nanolitre dispenser system (TTP LabTech, Melbourn, England) by mixing 75, 100 or 50 nl protein solutions with 75, 50 or 100 nl reservoir solutions, respectively. All drops were equilibrated against a 75 µl reservoir solution. A single crystal appeared in 2 d from a 1:1 ratio drop in condition No. 44 of Morpheus [the mixture of precipitants consisted of 12.5%(w/v) PEG 1000, 12.5%(w/v) PEG 3350 and 12.5%(v/v) MPD and the mixture of additives consisted of 0.02 M amino acids (0.2 M sodium L-glutamate, 0.2 M DL-alanine, 0.2 M glycine, 0.2 M DL-lysine–HCl, 0.2 M DL-serine) with buffer system 2 (0.1 M MOPS/HEPES–Na pH 7.5: a mixture of 26 ml 1 M MOPS with 24 ml 1 M HEPES–Na)]. Manual optimization of the crystal-growth conditions was performed using the hanging-drop vapour-diffusion method in 24-well VDX plates (Hampton Research, USA). The best crystals were grown at 293 K in drops consisting of a mixture of 1 µl protein solution and 1 µl reservoir solution; these drops were equilibrated against 200 µl reservoir solution.
A single crystal was flash-cooled directly in a liquid N2 stream at 100 K, as the mother liquor was an adequate cryoprotectant. The S-SAD data set (referred to as PfGrx1-SAD) was collected using Cu Kα radiation (λ = 1.54 Å) on a MAR 345 image-plate detector mounted on a Rigaku MicroMax-007 rotating-anode X-ray generator operated at 40 kV and 20 mA with VariMax HR optics. A total range of 360° was covered with 1.0° oscillation and 30 s exposure per frame. The crystal-to-detector distance was set to 100 mm. X-ray diffraction data were collected to 1.55 Å resolution. Other atomic resolution data sets (referred to as PfGrx1-AR1 and PfGrx1-AR2) were collected to 1.04 and 0.95 Å resolution using two different crystals (Supplementary Fig. S2) and two different wavelengths (0.83 and 0.73 Å) on BM14, ESRF, Grenoble. All data sets were indexed, processed, and scaled using the HKL-2000 package (Otwinowski & Minor, 1997). Bijvoet mates were treated as equivalent reflections during scaling but were merged separately as I+ and I− for the S-SAD data. Crystal parameters and data-processing statistics are summarized in Table 1.
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2.3. and refinement
PfGrx1 consists of 111 amino acids (∼12.4 kDa) with three methionines and three cysteines. From the crystal parameters, it was clear that there was one molecule per 3 Da−1 and a solvent content of 44%. The observed anomalous signal (〈ΔF〉/〈F〉) was 0.02, which was higher than the expected value of 0.01 at the Cu Kα radiation wavelength (λ = 1.54 Å) for a 111-residue protein with six S atoms (Hendrickson & Teeter, 1981; Dauter et al., 2002). The mean anomalous signal-to-noise ratio [〈ΔF/σ(ΔF)〉] was 1.47 and was significant to 2.0 Å resolution (Supplementary Table S1). Therefore, we chose the S-SAD approach to solve the A heavy-atom position search was performed to 2.0 Å resolution with SHELXD (Sheldrick, 2008), and the positions of six sulfur anomalous scatterers with occupancy >0.5 were obtained (Supplementary Fig. S3). These anomalous scatterers were further used to estimate the phases with SHELXE (Sheldrick, 2008). The electron-density maps generated by SHELXE had a clear, interpretable electron density, and 105 out of 111 polyalanine residues could be built in three chains (Fig. 1a). Furthermore, automatic model building was carried out with ARP/wARP (Langer et al., 2008) using the amino-acid sequence of PfGrx1, a polyalanine model, and the phases from SHELXE. The unit-cell parameters of the atomic resolution data sets (PfGrx1-AR1 and PfGrx1-AR2) were isomorphous to those for the PfGrx1-SAD data set. Therefore, atomic resolution structures were determined by 20 cycles of rigid-body using REFMAC5 (Murshudov et al., 2011) from the CCP4 suite (Winn et al., 2011) with the PfGrx1-SAD structure as a model Subsequently, a few runs of ten cycles of were carried out using REFMAC5. After each step of the models were inspected and manually adjusted to correspond to computed 2Fo − Fc and Fo − Fc electron-density maps using Coot (Emsley & Cowtan, 2004). During the progress of the 3-(N-morpholino)propanesulfonic acid (MOPS) and 2-methyl-2,4-pentanediol (MPD) solvent molecules and water molecules were manually added and included in the based on positive peaks in difference Fourier maps. The final round of was carried out using phenix.refine (Afonine et al., 2012). The occupancies of alternate conformations of protein residues and water molecules were also refined. The quality of the atomic model was assessed with PROCHECK (Laskowski et al., 1993) and MolProbity (Chen et al., 2010). The figures were generated using Chimera (Pettersen et al., 2004) and PyMOL (https://www.pymol.org ). Atomic coordinates and structure factors for the PfGrx1-SAD, PfGrx1-AR1 and PfGrx1-AR2 data sets have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, New Jersey, USA (https://www.rcsb.org/ ) with accession codes 4hjm , 4kje and 4kjf , respectively. The Friedel pairs for the S-SAD data were also included in the deposited structure factors.
with a Matthews coefficient of 2.21 Å3. Results and discussion
3.1. Model quality and overall description of PfGrx1
PfGrx1 has the typical glutaredoxin/thioredoxin fold with a core four-stranded β-sheet (strands β1 and β2 are parallel, and strands β3 and β4 are antiparallel) flanked by five α-helices (helices α1–α5; Fig. 1b). Very clear electron density was observed for all regions of the protein model except for the five N-terminal residues. SHELXD located an additional sulfur anomalous scatterer situated at the γGlu of the GSH-binding site. Based on electron-density maps, this site was identified as the S atom of a bound buffer solvent molecule: 3(N-morpholino)propanesulfonic acid (MOPS; Fig. 1c and Supplementary Fig. S4a). Additionally, a molecule of the crystallization precipitant 2-methyl-2,4-pentanediol (MPD) was found in the PfGrx1 structures (Fig. 1d and Supplementary Fig. S4b). The final and model parameters are summarized in Table 1. At atomic resolution, it is possible to refine alternate conformations of protein residues, bound ligands and water molecules (Dauter et al., 1995; Sekar et al., 2006; Liebschner et al., 2013). In all three PfGrx1 structures, 13–23 residues were modelled with alternate conformations, and most of these residues were polar/charged and solvent-exposed (Supplementary Table S2). Three buried residues (Ile21, Met48 and Arg78) were also modelled with alternate conformations. The orientations of the side chains were similar for most of the alternate conformations in all structures. The refined occupancy difference was less than 0.2 for the majority of the residues in all three structures. The r.m.s.d.s between the Cα atoms of the PfGrx1-SAD, PfGrx1-AR1 and PfGrx1-AR2 structures are insignificant (<0.07 Å). An analysis of Ramachandran plots calculated by MolProbity (Chen et al., 2010) showed that more than 99% of residues were in favoured regions, and the remainder (less than 1%) were in allowed regions.
3.2. The active-site and radiation-induced changes
The active-site cysteine pair containing the motif 29-CPYC-32 is located in a loop connecting the β1 strand to the α2 helix (Fig. 1b). The dithiol CPYC motif is identical to the active-site motif in ScGrx1 (PDB entries 3c1r and 3c1s ; Yu et al., 2008; Table 2), ScGrx2 (PDB entries 3ctf , 3ctg and 3d4m ; Discola et al., 2009; Li, Yu et al., 2010) and SmTGR (PDB entries 2v6o , 2x99 , 2x8c and 2x8g ; Angelucci et al., 2008, 2010). It is also very similar to CPFC motifs in EvGrx (PDB entries 2hze and 2hef ; Bacik & Hazes, 2007), SsGrx1 (PDB entry 1kte ; Katti et al., 1995) and HsGrx2 (PDB entries 2fls and 2ht9 ; Structural Genomics Consortium, unpublished work; Johansson et al., 2007). The continuous electron density between the S atoms of Cys29 and Cys32 shows an oxidized disulfide bond in PfGrx1-SAD and PfGrx-AR1 (Figs. 2a and 2b). The refined disulfide-bond lengths in PfGrx1-SAD and PfGrx1-AR1 are 2.05 and 2.10 Å, respectively; similar disulfide distances (2.06–2.08 Å) were observed in oxidized Grx structures (Katti et al., 1995; Yu et al., 2008; Bacik & Hazes, 2007; Discola et al., 2009; Li, Yu et al., 201) as well as in a survey of S—S distances [(2.04 (16) Å] in protein structures (Morris et al., 1992). The refined disulfide distance in PfGrx1-AR2 is 2.23 Å. Disulfide bond breakage can occur when using high-energy X-ray beams for diffraction data collection (Ravelli & McSweeney, 2000; Burmeister, 2000; Liebschner et al., 2013): S—S bond lengths first elongate and then break under the influence of absorbed X-rays. We therefore inspected difference electron-density maps around the disulfide bridge in the PfGrx1 structures to identify the likelihood of S—S bond breakage or alternate positions of Sγ atoms that are not connected to their partners. In PfGrx1-AR2, a strong negative density peak (8–10σ level) was observed at the Cys29–Cys32 bridge, whereas in PfGrx1-AR1 a negative peak (<6σ level) was only observed for the Sγ atom of Cys29. In PfGrx1-AR2, a positive peak (6–8σ level) was observed near Cys29 so that an alternate position of the Sγ atom could be located. The positive peak near Cys29 in PfGrx1-AR1 is significantly weaker (<4σ level), and no alternate position could be located. The refined occupancies in the A and B conformations are 0.65 and 0.35, respectively. The major conformation of Cys29 was still connected to its partner Cys32. The normalized χ2 values indicate that the PfGrx1-AR2 data set (χ2 = 1.131) suffered more radiation damage than the PfGrx1-AR1 data set (χ2 = 0.839). The deposited radiation dose was calculated using RADDOSE (Zeldin et al., 2013). The average and maximum doses deposited were 6.9 and 67.9 kGy, respectively, for the PfGrx1-AR1 crystals and 7.9 and 78.6 kGy, respectively, for the PfGrx1-AR2 crystals. Therefore, radiation damage may be limited. The average B factor of the active-site CPYC motif in the PfGrx1 structures (14.4, 8.4 and 8.5 Å2 for PfGrx1-SAD, PfGrx1-AR1 and PfGrx1-AR2, respectively) is lower than the overall B values (Table 1), which are similar to those of other dithiol Grxs (Rouhier et al., 2007; Yu et al., 2008; Discola et al., 2009; Li, Yu et al., 2010).
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3.3. Structural comparison of PfGrx1 with other Grxs/Grx-like domains
The sequence identity between Grxs and Grx-like domains is in the range 16–37% and few residues (<15) are conserved among them (Fig. 3a and Table 2). The superposition of 74–104 Cα atoms of the PfGrx1 structure with monothiol/dithiol, oxidized/reduced and GSH-bound/unbound forms of Grx structures from different organisms shows an r.m.s.d. range of 1.3–2.3 Å (Fig. 3b and Table 2), suggesting that the overall folding of PfGrx1 is similar to that of other Grxs and Grx-like domains. Superposition of Grxs and Grx-like domains shows very good agreement for the core β-strands and the active-site motif containing the α2 helix and the α4 helix (Fig. 3b). Apart from the insertion loops, the maximum displacement was observed in the α1, α3 and α5 helices. Most conserved residues are present around the active-site regions of these enzymes (Fig. 3b). The GSH-binding motifs (CXXC or CXXS, TVP and CDD) are structurally conserved in oxidized/reduced and GSH-bound/unbound forms of both the monothiol and dithiol Grxs except for the Gly-binding residue at position 63 (Fig. 3c). In most Grxs, a conserved hydrophobic residue followed by a Gly-Gly doublet precedes the CDD motif. The Gly-Gly doublet provides room for two conserved water molecules (Wc1 and Wc2) which interact with the γGlu of GSH. Bound GSH molecules adopt extended conformations apart from in FtGrx (PDB entry 3msz ; Fig. 3c). In all Grxs, highly positively charged surfaces were observed in the GSH chelating regions; however, distinct surfaces were observed between the Gly- and γGlu-binding sites (Fig. 4). The basic charge congregations are owing to the conserved residues Lys and Gln/Arg at positions 26 (based on the PfGrx1 residue numbering) and 63, respectively. The distinct potential distribution in the γGlu-binding region is a consequence of fewer conserved residues in the CDD motif as well as at positions 72 (Lys/Arg/Gln/Trp) and 77 (Arg/Gln/Asn) (Fig. 3a).
3.4. GSH-binding site in PfGrx1
The solvent-exposed GSH-binding channel in PfGrx1 is filled with several water molecules, and the γGlu portion of the GSH-binding pocket is occupied by MOPS (Figs. 1c and 5a, and Supplementary Figs. S3 and S4a). Bound MOPS interacts with the CDD and TVP motifs. The B factor of the bound MOPS molecule is lower than that of the protein atoms in all three PfGrx1 structures (Table 1). Our co-crystallization and cryo-soaking attempts failed to provide crystals of the PfGrx1–GSH complex. This may be owing to a higher binding affinity of PfGrx1 for buffer molecules (Fig. 5a). Therefore, we modelled the GSH molecule into the PfGrx1 active site by superimposing GSH-bound HsGrx2 (PDB entry 2fls ; Structural Genomics Consortium, unpublished work) onto PfGrx1 (Fig. 5b). The backbone carboxyl O and amide N atoms of Cys and γGly in GSH invariantly interact with the backbone N and O atoms of the conserved TVP (Thr-Val-Pro) and CDD (Cys-Asp-Asp) motifs. In the present atomic resolution PfGrx1 structures, six GSH binding pocket residues (Lys26, Cys29, Pro30, Lys72, Arg77 and Asp90) adopt alternate conformations. In PfGrx1, polygonal water structures such as tetragons and pentagons were observed (Fig. 5a and Supplementary Fig. S4a). Hydrogen-bonding interactions between Lys26 and Gln63 are absent in PfGrx1 when compared with other GSH-bound and free Grxs (ScGrx1 and ScGrx2; PDB entries 3c1r , 3c1s , 3ctf , 3ctg and 3d4m ; Yu et al., 2008; Li, Yu et al., 2010; Discola et al., 2009).
3.5. Conserved water molecules and unique features of PfGrx1
Three conserved water molecules (Wc1, Wc2 and Wc3) are observed for most Grxs (Figs. 3c and 5c). Wc1 and Wc2 are located at the GSH-binding site and are involved in hydrogen-bonding interactions between the GSH molecule and the TVP and CDD motifs. The other conserved water molecule Wc3 is located at the position of the α1 helix, the β1 strand, and the loop between the β3 and β4 strands. In PfGrx1, Glu28 precedes the active-site residues, and the corresponding position in other Grxs is populated by polar Ser/Thr/Asn, aromatic Tyr/Trp, or Gly residues. Similarly, Glu51 is located between the α1 helix and the β2 strand, and the corresponding position in other Grxs has a conserved hydrophobic residue Val/Leu/Ile or aromatic residue Phe/Tyr (Fig. 3a). A water-mediated salt bridge between the α1 helix and the loop between the β3 and β4 strands is found in PfGrx1, and this interaction is absent in other Grxs (Fig. 5c). Unique salt bridges between the α1 and α3 helices and the β4 strand and the α4 and α5 helices were found only in PfGrx1, PtGrxS12 and SsGrx1 (Supplementary Fig. S5).
4. Conclusions
In this work, we have determined the atomic structure of the dithiol Grx PfGrx1 and compared it in depth with those of other Grxs from different organisms. Our results indicate that monothiol (CXXS) and dithiol (CXXC) Grxs differ significantly in their helix-capping hydrogen bonds (Supplementary Figs. S6, S7 and S8). Both monothiol and dithiol Grxs contain three conserved water molecules, of which two are located in the GSH-binding site while the third is located between β-strands and the α1 helix. The dithiol-containing redox proteins thioredoxin (Trx), glutaredoxin (Grx) and plasmoredoxin (Plrx), with the latter being exclusively found in Plasmodium species, play central roles in maintaining redox homeostasis in malarial parasites (Sturm et al., 2009). The PfGrx1 structure presented here in complex with MOPS and MPD provides novel insights concerning interacting surfaces whose roles in in vivo interactions with parasite biomolecules remain to be explored in further detail.
Supporting information
3D view: 4hjm,4kje,4kjf
PDB references: glutaredoxin 1, 4hjm; 4kje; 4kjf
Supplementary material. DOI: 10.1107/S1399004713025285/rr5049sup1.pdf
Acknowledgements
The X-ray facility at ICGEB was funded by the Wellcome Trust. This work was generously supported with grants from the Department of Biotechnology (DBT), Government of India to TT, AS and MY, and with the Deutsche Forschungsgemeinschaft (BE 1540/18-1 to KB and SR).
References
Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Angelucci, F., Dimastrogiovanni, D., Boumis, G., Brunori, M., Miele, A. E., Saccoccia, F. & Bellelli, A. (2010). J. Biol. Chem. 285, 32557–32567. Web of Science CrossRef CAS PubMed Google Scholar
Angelucci, F., Miele, A. E., Boumis, G., Dimastrogiavanni, D., Brunori, M. & Belleli, A. (2008). Proteins, 72, 936–945. Web of Science CrossRef PubMed CAS Google Scholar
Axelsson, K. & Mannervik, B. (1980). Biochim. Biophys. Acta, 613, 324–336. CrossRef CAS PubMed Web of Science Google Scholar
Bacik, J. P. & Hazes, B. (2007). J. Mol. Biol. 365, 1545–1558. Web of Science CrossRef PubMed CAS Google Scholar
Balmer, Y., Koller, A., del Val, G., Manieri, W., Schürmann, P. & Buchanan, B. B. (2003). Proc. Natl Acad. Sci. USA, 100, 370–375. Web of Science CrossRef PubMed CAS Google Scholar
Berndt, C., Lillig, C. H. & Holmgren, A. (2008). Biochim. Biophys. Acta, 1783, 641–650. Web of Science CrossRef PubMed CAS Google Scholar
Burmeister, W. P. (2000). Acta Cryst. D56, 328–341. Web of Science CrossRef CAS IUCr Journals Google Scholar
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21. Web of Science CrossRef CAS IUCr Journals Google Scholar
Chrestensen, C. A., Starke, D. W. & Mieyal, J. J. (2000). J. Biol. Chem. 275, 26556–26565. Web of Science CrossRef PubMed CAS Google Scholar
Couturier, J., Koh, C. S., Zaffagnini, M., Winger, A. M., Gualberto, J. M., Corbier, C., Decottignies, P., Jacquot, J.-P., Lemaire, S. D., Didierjean, C. & Rouhier, N. (2009). J. Biol. Chem. 284, 9299–9310. Web of Science CrossRef PubMed CAS Google Scholar
Couturier, J., Ströher, E., Albetel, A. N., Roret, T., Muthuramalingam, M., Tarrago, L., Seidel, T., Tsan, P., Jacquot, J.-P., Johnson, M. K., Dietz, K. J., Didierjean, C. & Rouhier, N. (2011). J. Biol. Chem. 286, 27515–27527. Web of Science CrossRef CAS PubMed Google Scholar
Dauter, Z., Dauter, M. & Dodson, E. J. (2002). Acta Cryst. D58, 494–506. Web of Science CrossRef CAS IUCr Journals Google Scholar
Dauter, Z., Lamzin, V. S. & Wilson, K. S. (1995). Curr. Opin. Struct. Biol. 5, 784–790. CrossRef CAS PubMed Web of Science Google Scholar
Discola, K. F., de Oliveira, M. A., Rosa Cussiol, J. R., Monteiro, G., Bárcena, J. A., Porras, P., Padilla, C. A., Guimarães, B. G. & Netto, L. E. (2009). J. Mol. Biol. 385, 889–901. Web of Science CrossRef PubMed CAS Google Scholar
Djuika, C. F., Fiedler, S., Schnölzer, M., Sanchez, C., Lanzer, M. & Deponte, M. (2013). Biochim. Biophys. Acta, 1830, 4073–4090. Web of Science CrossRef CAS PubMed Google Scholar
Eklund, H., Ingelman, M., Söderberg, B. O., Uhlin, T., Nordlund, P., Nikkola, M., Sonnerstam, U., Joelson, T. & Petratos, K. (1992). J. Mol. Biol. 228, 596–618. CrossRef PubMed CAS Web of Science Google Scholar
Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132. Web of Science CrossRef CAS IUCr Journals Google Scholar
Feng, Y., Zhong, N., Rouhier, N., Hase, T., Kusunoki, M., Jacquot, J.-P., Jin, C. & Xia, B. (2006). Biochemistry, 45, 7998–8008. Web of Science CrossRef PubMed CAS Google Scholar
Fernandes, A. P. & Holmgren, A. (2004). Antioxid. Redox Signal. 6, 63–74. Web of Science CrossRef PubMed CAS Google Scholar
Gallogly, M. M., Starke, D. W. & Mieyal, J. J. (2009). Antioxid. Redox Signal. 11, 1059–1081. Web of Science CrossRef PubMed CAS Google Scholar
Gonzalez Porqué, P., Baldesten, A. & Reichard, P. (1970). J. Biol. Chem. 245, 2371–2374. PubMed Web of Science Google Scholar
Hendrickson, W. A. & Teeter, M. M. (1981). Nature (London), 290, 107–113. CrossRef CAS Web of Science Google Scholar
Hoff, K. G., Culler, S. J., Nguyen, P. Q., McGuire, R. M., Silberg, J. J. & Smolke, C. D. (2009). Chem. Biol. 16, 1299–1308. Web of Science CrossRef CAS PubMed Google Scholar
Holmgren, A. (1976). Proc. Natl Acad. Sci. USA, 73, 2275–2279. CrossRef CAS PubMed Web of Science Google Scholar
Holmgren, A. (1979). J. Biol. Chem. 254, 3672–3678. CAS PubMed Web of Science Google Scholar
Holmgren, A. (2000). Antioxid. Redox Signal. 2, 811–820. Web of Science CrossRef PubMed CAS Google Scholar
Holmgren, A., Johansson, C., Berndt, C., Lönn, M. E., Hudemann, C. & Lillig, C. H. (2005). Biochem. Soc. Trans. 33, 1375–1377. Web of Science PubMed CAS Google Scholar
Iwema, T., Picciocchi, A., Traore, D. A. K., Ferrer, J.-L., Chauvat, F. & Jacquamet, L. (2009). Biochemistry, 48, 6041–6043. Web of Science CrossRef PubMed CAS Google Scholar
Johansson, C., Kavanagh, K. L., Gileadi, O. & Oppermann, U. (2007). J. Biol. Chem. 282, 3077–3082. Web of Science CrossRef PubMed CAS Google Scholar
Johansson, C., Roos, A. K., Montano, S. J., Sengupta, R., Filippakopoulos, P., Guo, K., von Delft, F., Holmgren, A., Oppermann, U. & Kavanagh, K. L. (2011). Biochem. J. 433, 303–311. Web of Science CrossRef CAS PubMed Google Scholar
Katti, S. K., Robbins, A. H., Yang, Y. & Wells, W. W. (1995). Protein Sci. 4, 1998–2005. CrossRef CAS PubMed Google Scholar
Kehr, S., Sturm, N., Rahlfs, S., Przyborski, J. M. & Becker, K. (2010). PLoS Pathog. 6, e1001242. Web of Science CrossRef PubMed Google Scholar
Kim, S. G., Chung, J.-S., Sutton, R. B., Lee, J.-S., López-Maury, L., Lee, S. Y., Florencio, F. J., Lin, T., Zabet-Moghaddam, M., Wood, M. J., Nayak, K., Madem, V., Tripathy, J. N., Kim, S.-K. & Knaff, D. B. (2012). Biochim. Biophys. Acta, 1824, 392–403. Web of Science CrossRef CAS PubMed Google Scholar
Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. (2008). Nature Protoc. 3, 1171–1179. Web of Science CrossRef CAS Google Scholar
Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). J. Appl. Cryst. 26, 283–291. CrossRef CAS Web of Science IUCr Journals Google Scholar
Lemaire, S. D., Guillon, B., Le Maréchal, P., Keryer, E., Miginiac-Maslow, M. & Decottignies, P. (2004). Proc. Natl Acad. Sci. USA, 101, 7475–7480. Web of Science CrossRef PubMed CAS Google Scholar
Li, L., Cheng, N., Hirschi, K. D. & Wang, X. (2010). Acta Cryst. D66, 725–732. Web of Science CrossRef CAS IUCr Journals Google Scholar
Li, W.-F., Yu, J., Ma, X.-X., Teng, Y.-B., Luo, M., Tang, Y.-J. & Zhou, C.-Z. (2010). Biochem. Biophys. Acta, 1804, 1542–1547. Web of Science CrossRef CAS PubMed Google Scholar
Liebschner, D., Dauter, M., Brzuszkiewicz, A. & Dauter, Z. (2013). Acta Cryst. D69, 1447–1462. Web of Science CrossRef CAS IUCr Journals Google Scholar
Lill, R., Dutkiewicz, R., Elsässer, H. P., Hausmann, A., Netz, D. J., Pierik, A. J., Stehling, O., Urzica, E. & Mühlenhoff, U. (2006). Biochim. Biophys. Acta, 1763, 652–667. Web of Science CrossRef PubMed CAS Google Scholar
Lillig, C. H., Berndt, C. & Holmgren, A. (2008). Biochim. Biophys. Acta, 1780, 1304–1317. Web of Science CrossRef PubMed CAS Google Scholar
Lillig, C. H., Berndt, C., Vergnolle, O., Lönn, M. E., Hudemann, C., Bill, E. & Holmgren, A. (2005). Proc. Natl Acad. Sci. USA, 102, 8168–8173. Web of Science CrossRef PubMed CAS Google Scholar
Lindahl, M. & Florencio, F. J. (2003). Proc. Natl Acad. Sci. USA, 100, 16107–16112. Web of Science CrossRef PubMed CAS Google Scholar
Luo, M., Jiang, Y.-L., Ma, X.-X., Tang, Y.-J., He, Y.-X., Yu, J., Zhang, R.-G., Chen, Y. & Zhou, C.-Z. (2010). J. Mol. Biol. 398, 614–622. Web of Science CrossRef CAS PubMed Google Scholar
Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J. & Hay, R. T. (1992). Nucleic Acids Res. 20, 3821–3830. CrossRef PubMed CAS Web of Science Google Scholar
Morris, A. L., MacArthur, M. W., Hutchinson, E. G. & Thornton, J. M. (1992). Proteins, 12, 345–364. CrossRef PubMed CAS Web of Science Google Scholar
Motohashi, K., Kondoh, A., Stumpp, M. T. & Hisabori, T. (2001). Proc. Natl Acad. Sci. USA, 98, 11224–11229. Web of Science CrossRef PubMed CAS Google Scholar
Mühlenhoff, U., Molik, S., Godoy, J. R., Uzarska, M. A., Richter, N., Seubert, A., Zhang, Y., Stubbe, J., Pierrel, F., Herrero, E., Lillig, C. H. & Lill, R. (2010). Cell Metab. 12, 373–385. Web of Science PubMed Google Scholar
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. CrossRef CAS Web of Science Google Scholar
Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. & Ferrin, T. E. (2004). J. Comput. Chem. 25, 1605–1612. Web of Science CrossRef PubMed CAS Google Scholar
Pineda-Molina, E., Klatt, P., Vázquez, J., Marina, A., García de Lacoba, M., Pérez-Sala, D. & Lamas, S. (2001). Biochemistry, 40, 14134–14142. Web of Science PubMed CAS Google Scholar
Rahlfs, S., Fischer, M. & Becker, K. (2001). J. Biol. Chem. 276, 37133–37140. Web of Science CrossRef PubMed CAS Google Scholar
Ravelli, R. B. G. & McSweeney, S. M. (2000). Structure, 8, 315–328. Web of Science CrossRef PubMed CAS Google Scholar
Rouhier, N., Unno, H., Bandyopadhyay, S., Masip, L., Kim, S.-K., Hirasawa, M., Gualberto, J. M., Lattard, V., Kusunoki, M., Knaff, D. B., Georgiou, G., Hase, T., Johnson, M. K. & Jacquot, J.-P. (2007). Proc. Natl Acad. Sci. USA, 104, 7379–7384. Web of Science CrossRef PubMed CAS Google Scholar
Rouhier, N., Villarejo, A., Srivastava, M., Gelhaye, E., Keech, O., Droux, M., Finkemeier, I., Samuelsson, G., Dietz, K. J., Jacquot, J.-P. & Wingsle, G. (2005). Antioxid. Redox Signal. 7, 919–929. Web of Science CrossRef PubMed CAS Google Scholar
Sekar, K., Yogavel, M., Gayathri, D., Velmurugan, D., Krishna, R., Poi, M.-J., Dauter, Z., Dauter, M. & Tsai, M.-D. (2006). Acta Cryst. F62, 1–5. Web of Science CrossRef CAS IUCr Journals Google Scholar
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Web of Science CrossRef CAS IUCr Journals Google Scholar
Shelton, M. D., Chock, P. B. & Mieyal, J. J. (2005). Antioxid. Redox Signal. 7, 348–366. Web of Science CrossRef PubMed CAS Google Scholar
Sturm, N., Jortzik, E., Mailu, B. M., Koncarevic, S., Deponte, M., Forchhammer, K., Rahlfs, S. & Becker, K. (2009). PLoS Pathog. 5, e1000383. Web of Science CrossRef PubMed Google Scholar
Tripathi, T., Röseler, A., Rahlfs, S., Becker, K. & Bhakuni, V. (2010). Biochimie, 92, 284–291. Web of Science CrossRef CAS PubMed Google Scholar
Tsang, M. L. (1981). J. Bacteriol. 146, 1059–1066. CAS PubMed Web of Science Google Scholar
Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Web of Science CrossRef CAS IUCr Journals Google Scholar
Wong, J. H., Cai, N., Balmer, Y., Tanaka, C. K., Vensel, W. H., Hurkman, W. J. & Buchanan, B. B. (2004). Phytochemistry, 65, 1629–1640. Web of Science CrossRef PubMed CAS Google Scholar
Yu, J., Zhang, N.-N., Yin, P.-D., Cui, P.-X. & Zhou, C.-Z. (2008). Proteins, 72, 1077–1083. Web of Science CrossRef PubMed CAS Google Scholar
Zeldin, O. B., Gerstel, M. & Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230. Web of Science CrossRef CAS IUCr Journals Google Scholar
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