Glutathionylation‐triggered conformational changes of glutaredoxin Grx1 from the yeast Saccharomyces cerevisiae (2008)

Glutaredoxins are thermo-stable thiol-disulfide oxidoreductases with different cellular functions, especially important in the cellular redox homeostasis. They are first identified as electron donors for ribonucleotide reductases,1 and also involved in a series of essential biosynthetic reactions and regulation in many biological processes as a glutathione-dependent manner.1-3 Glutaredoxins, coupled with NADPH, GSH, and glutathione reductase, consist of the glutaredoxin system, which transfers electrons from NADPH to glutaredoxins via GSH,4 protecting cells from damages caused by reactive oxygen species (ROS) during the course of normal aerobic metabolism or following exposure to radical-generating compounds, especially when the cellular survival mechanisms are unable to cope with the ROS and/or resulted damages.5, 6 Structurally glutaredoxins (Grxs) belong to the Trx-like fold family, characterized with a β-pleated sheet at the center of the molecule surrounded by five helices and a dithiol active site CXXC motif, despite that some members in Grxs are monothiolic.3, 7-11 Many studies have highlighted the key roles played by sulfhydryl groups (SH) in response to oxidative stress, and particularly, the roles of the GSH/Grxs and thioredoxin (Trx) systems in maintaining the cellular redox homeostasis.12-15 To date five glutaredoxins (Grx1-5) have been identified in yeast S. cerevisiae and classified into two subfamilies. Grx1 and Grx2 belong to the dithiol glutaredoxin subfamily with the active site CPYC motif16 and Grx3-5 belong to the monothiol glutaredoxin subfamily with the active site CGFS motif.9 In dithiolic glutaredoxins the N-terminal cysteine commonly has a relatively low pKa and exists as thiolate in solution, which is important to thiol-disulfide exchange reaction.17-20 The thiolate initializes nucleophilic attack on GSH-mixed disulfide to form a Grx-substrate intermediate, which can be reduced by the C-terminal cysteine, causing the formation of disulfide between two cysteines and the releasing of reduced substrate. Although a lot of glutaredoxin structures from E. coli, poxvirus, pig, and human have been reported, yeast Grx1 shares only 37% highest sequence identity with these structures. The crystal structure of GSH bound Grx1-C30S mutant has been reported recently (PDB code 2JAC), however the distance between sulfur atoms of GSH and Cys27 is 2.88 Å, far beyond the normal disulfide bond length.21 The structure of glutathionylated Grx1 in our work has a standard length of disulfide bond and the side-chain orientation of Cys27 is different from that of 2JAC. Moreover comparative structural analyses of the oxidized and glutathionylated Grx1 illustrate the conformational changes of residues at different redox states, whose roles during catalysis are further compared with the homologous structures. The GRX1/YCL035C open reading frame was amplified by PCR, using S. cerevisiae S288c genomic DNA as the template, and cloned into a pET28b-derived plasmid (Novagen). Extra sequence encoding MGHHHHHH was added at the N-terminal. The recombinant protein was overexpressed by using Rosetta (DE3) strain of Escherichia coli induced with 0.2 mM IPTG at 37°C for 5 h when OD600 nm reached 0.6. Pellets were collected by centrifugation at 8000g for 10 min at 4°C and re-suspended in 200 mM NaCl, 20 mM Tris-HCl, pH 7.0, and followed by sonication. After centrifugation at 25,000g for 30 min at 4°C, the supernatant was collected and applied to a Ni2+-NTA column (Amersham Biosciences). The target protein was eluted with 300 mM imidazole in 200 mM NaCl, 20 mM Tris-HCl, pH 7.0, and loaded to a Superdex 75 16/60 column, equilibrated with 200 mM NaCl, 20 mM Tris-HCl, pH 7.0. Fractions containing the recombinant Grx1 were pooled, desalted, and concentrated to 10 mg/mL in a final buffer of 20 mM NaCl, 20 mM Tris-HCl, pH 7.0. Protein concentration was determined by its absorbance at 280 nm with a theoretical molar extinction coefficient of 6085 M−1 cm−1 (http://www.expasy.org). To check the remaining free thiols of different protein samples, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) assay was performed as previously described.22 Briefly, the protein sample was diluted with the reaction buffer (0.1M sodium phosphate, pH 8.0) to a final volume of 250 μL, and mixed with 50 μL of Ellman's reagent solution (4 mg DTNB in 1 mL of reaction buffer), then incubated at 25°C for 15 min. The absorbance at 412 nm was measured to determine the sulfhydryl content with a molar extinction of 14,150 M−1 cm−1. As a blank control, 250 μL of reaction buffer without protein was used. Crystals of oxidized and glutathionylated Grx1 were grown at 16°C in a hanging drop of 1.0 μL protein sample at 10 mg/mL in 20 mM Tris-HCl, 20 mM NaCl, pH 7.0 with equal volume of reservoir solution. Oxidized Grx1 was crystallized in 12% PEG 20,000 and 0.1M MES (2-(N-morpholino)ethanesulfonic acid), pH 6.5. Glutathionylated Grx1 was cocrystallized with 20 mM GSH in 20% PEG 4000, 0.1M sodium acetate, pH 5.3, and 0.2M ammonium dihydrogen phosphate. The crystal was transferred to the cryoprotectant of the reservoir solution supplemented with 25% glycerol and flash cooled with liquid nitrogen. The X-ray diffraction data were collected at 100 K in a liquid nitrogen stream using a Rigaku MM007 X-ray generator (λ = 1.5418 Å) with a MarResearch 345 image-plate detector at School of Life Sciences, University of Science and Technology of China (USTC, Hefei, China). Data were processed using AUTOMAR 1.4.23 The structure of oxidized form was solved by the molecular replacement method with the program CNS24 using the atomic coordinates of human Grx2 complexed with GSH (PDB code 2FLS) as the search model (36% sequence identity). The structure of glutathionylated Grx1 was solved using the oxidized Grx1 structure as the model. Both structures were fitted and rebuilt with program O25 and refined with REFMAC526 and CNS.24 The overall assessments of model quality were performed using PROCHECK V 3.4.4.27 All figures were prepared with PyMOL.28 According to the results of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) assay, the recombinant Grx1 overexpressed in E. coli was purified in oxidized form without glutathionylation (data not shown). Statistics of Grx1 structures are summarized in Table I. The key residues are highly conserved among all glutaredoxins (see Fig. 1). Similar to its homologues, the overall structure of Grx1 is consistent with the glutaredoxin fold (Trx-like fold), with a core of four mixed β-strands, in which three are antiparallel (β1, β3, β4) with strand β2 parallel to the adjacent β1 [Fig. 2(A)]. The central β-sheet is surrounded by five α-helices, which is similar to crystal structures of pig Grx1 (PDB code 1KTE) and human Grx2 (PDB code 2FLS), while helices α1 and α5 are absent from E. coli Grx1 (PDB code 1EGO) or Grx3 (PDB code 3GRX). Two η-helices are also assigned on each side of strand β2 in Grx1 while the η-helix before strand β2 is missing in E. coli Grx3, human Grx2, and poplar Grx-C1 (PDB code 1Z7R), due to the lack of corresponding residues (see Fig. 1). Sequence multi-alignment of Grx1 and its homologues. The alignment was performed using MultAlin and ESPript (http://prodes.toulouse.inra.fr/multalin).29, 30 The secondary structural elements are identified from structures of S. cerevisiae and poxviral (PDB code: 2HZF) and displayed at the top and bottom of the alignment, respectively. The α-helices, η-helices, β-sheets, and strict β-turns are denoted as, α, η, β and TT, correspondingly. Completely conserved residues are indicated by white lettering on a red background. Residues involved in binding of GSH are labeled with stars below the sequence. (A) Cartoon representation of the overall structure of glutathionylated Grx1. Secondary structures are labeled with the α-helix and η-helix colored in cyan and β-strand in purple. (B) The omit map of glutathionylated Grx1. Contours are drawn at 1.5 σ. (C) Interactions between GSH and Grx1 in glutathionylated Grx1. C atoms in GSH are colored in yellow, while C atoms in Grx1 are colored in palecyan. Other atoms are colored according to atom types, S in gold, O in red, and N in blue. Water molecules are illustrated as red dots. Distances between atoms are labeled in Å. In the structure of glutathionylated Grx1, a GSH molecule forms disulfide bond with Cys27 and the distance between sulfur atoms of GSH and Cys27 is refined to 2.10 Å, as shown in the omit map [Fig. 2(B)]. Surface analyses illustrated a well-defined GSH binding groove in Grx1, similar to other GSH-complexed glutaredoxin structures.31, 32 The groove is composed of residues which are mostly conserved in glutaredoxins, labeled with stars in Figure 1. Comparisons of surface and electrostatic potentials in these structures demonstrate that the groove is specifically GSH-shaped and nearly positively charged, finely complementary to the shape and charge of GSH molecule. This groove does not exist in Trx, DsbA, and PDI, consistent with their lack of GSH-binding capacity.2, 33 Sequence multi-alignment shows this groove mainly consists of two regions, Ile85-Asp89 and Thr74-Pro76, and a patch composing of Lys24 and Gln63, contributing to the recognition and stabilization of the three moieties of GSH molecule. In the first GSH binding region, residues Asn88 and Asp89 form intensive hydrogen bonds with the γ-glutamyl moiety of GSH. Residues Ile85, Gly86, and Gly87 assist in the formation and orientation of helix α4, which is crucial for the proper positioning of Asn88 and Asp89 to stabilize GSH. Comparing with glutathionylated E. coli Grx3 and human Grx1, the conformation of γ-glutamyl moiety of GSH in glutathionylated Grx1 adopts a different conformation with highly favorable interactions of its two carboxyl oxygen atoms and amide nitrogen atom to hydrogen bond with amide nitrogen atoms of Asn88, Asp89 and carboxyl at side chain of Asp89, respectively [Fig. 2(C)]. Contrasted to the oxidized Grx1, the bonded GSH in glutathionylated Grx1 causes the side chain hydroxyl of Tyr29 to move 1.61 Å towards GSH, despite that the oxidized form has inserted with one MES molecule similar to the γ-glutamyl moiety of GSH. The distance between the γ-glutamyl moiety of GSH and side chain of Tyr29 [Fig. 2(C)] is within 6 Å, resulting in very strong hydrophobic interactions34 with GSH. These interactions could be found from other glutathionylated (PDB code 3GRX and 1B4Q) and GSH-bound glutaredoxins (PDB code 2FLS and 2JAC). Furthermore, according to the glutaredoxin active site pattern search in PROSITE (http://www.expasy.org/prosite/), the second X in CXXC motif corresponds to either one of residues of Phe, Tyr, Trp, and His, which addresses the importance of hydrophobic interactions in stabilizing the approaching GSH. The second GSH binding region consists of residues Thr74, Val75, and Pro76. In the glutathionylated Grx1, one of the oxygen atoms of glutamyl moiety in GSH forms two water-mediated hydrogen bonds with the hydroxyl of Thr74 and carbonyl oxygen of Pro76, coupling with the carbonyl oxygen and amide nitrogen of cysteinyl group in GSH-forming hydrogen bonds with amide nitrogen and carbonyl oxygen of Val75 [Fig. 2(C)]. Such hydrogen bond interactions are also found in other GSH complex structures of E. coli Grx1 and Grx3 and human Grx1 and Grx2. Meanwhile, residue Pro76 in yeast Grx1 exists in the cis conformation to introduce a sharp turn at the start of strand β3, which helps Grx1 to adapt the γ-glutamyl moiety of GSH specifically. A recent paper indicates that the γ-linkage between γ-glutamine and cysteine of GSH, which stacks on Pro76, is important in the specificity of GSH to the GSH-shaped groove in E. coli Grx1, demonstrated by the absence of mixed disulfide product between E. coli Grx1 and GSH analog, glutamylcysteinylglycine.35 Residues Lys24 and Gln63 are contributing to binding the glycine moiety of GSH, as observed in other GSH-complexed glutaredoxins.36 The conserved Gln63 forms a hydrogen bond network with the highly conserved Lys24, the carboxylate of glycine moiety of GSH, and the main chain carbonyl oxygen of Arg73 [Fig. 2(C)]. Gln63 in yeast Grx1 is corresponding to Arg58 in poxviral glutaredoxin. Although in poxviral glutaredoxin the electrostatic repulsion will drive both positively charged residues of Lys20 and Arg58 apart, it is proposed that Arg58 could adopt a conformation to make a salt bridge with the carboxylate of glycine moiety in GSH thus avoid the electrostatic conflict with Lys20,37 as Arg67 and Arg40 in solution structures of glutathionylated human Grx131 and E. coli Grx3,32 respectively. In the oxidized yeast Grx1, B-factors of the region Lys24-Tyr26 are almost twice to those of the adjacent residues, which is also observed at corresponding region either in oxidized pig Grx1 or both oxidized and reduced poxviral glutaredoxin. Moreover in structures of glutathionylated yeast Grx1 and human Grx2 (PDB code 2FLS), B-factors in this region decrease to the same level as neighboring residues (data not shown). This phenomenon demonstrates that this region, either in the oxidized form or reduced form, is relatively flexible to be ready for GSH or disulfide substrates binding, which can be also found in solution structures of E. coli Grx1 (PDB code 1EGO, 1EGR, 1GRX, and 1QFN) and Grx3 (PDB code 3GRX). Subsequent to the break of disulfide between Cys27 and Cys30 in glutathionylated Grx1, dramatic conformational rearrangements were found at residues of both Thr25 and Tyr26 [Fig. 3(A)]. The orientation of carbonyl oxygen atom of Thr25 flips reversely, causing main chain C atom to move with a distance of 0.92 Å in glutathionylated Grx1, accompanied by the reorientation of carbonyl oxygen atom of Tyr26. Interestingly the aromatic side chain of Tyr26 is found flipping around towards the active site in the glutathionylated form, causing the side chain hydroxyl of Tyr26 to move 12.22 Å and form one water-mediated hydrogen bond with the carboxylate oxygen atom of glycine moiety in GSH. Residues Thr25 and Tyr26 in Grx1 are corresponding to Tyr21 and Thr22 in poxviral glutaredoxin, respectively. In the reduced poxviral glutaredoxin, the side chain of Tyr21 is packed onto a strongly hydrophobic pocket while solvent-exposed in oxidized state; the main chain oxygen atom of Thr22 moving towards the solvent and its side chain tilted towards the active site, which is similar to Tyr26 in yeast Grx1 [Fig. 3(B)]. We have also soaked the oxidized Grx1 crystal into the reservoir solution containing 20 mM DTT, and determined its structure as an artificial reduced state with the break of disulfide between Cys27 and Cys30. To our surprise, although the side-chain of Tyr26 is fixed in the reduced yeast Grx1 structure as in the oxidized form due to crystal packing, the reorientation of main chain atoms of Thr25 and Tyr26 is completely consistent with the glutathionylated Grx1 and reduced poxviral glutaredoxin. It indicated that the conformation of main chain atoms of Thr25 and Tyr26 is controlled by the disulfide bond between Cys27 and Cys30, whose breakage will trigger the reorientation of Thr25 and Tyr26, followed by flipping of the side chain of Tyr26. (A) Stereoview of conformational changes from Lys24 to His31 in oxidized and glutathionylated Grx1, of which C atom is colored in yellow and palecyan, respectively. Other atoms are colored as Figure 2. (B) Stereoview of structural superposition between glutathionylated Grx1 and reduced poxviral glutaredoxin, with C atom colored in palecyan and green, respectively. Residues in yeast Grx1 are labeled and corresponding residues in poxviral glutaredoxin are label in parentheses. In addition, contrasted to the oxidized Grx1, the side chain of His31 turns around about 120° in glutathionylated Grx1, stacking on the Cys27-Pro28 dipeptide [Fig. 2(A)]. The Nε2 atom in the side chain of His31 forms a hydrogen bond with the carbonyl oxygen atom of Thr25 at a length of 2.69 Å. Such a conformational change has been mentioned in the study of pKa value of N-terminal cysteine in reduced pig glutaredoxin,38 in which Arg26, corresponding to His31 in yeast Grx1, can swing to stack on the CPFC motif and form hydrogen bonds with carbonyl oxygen atoms of Pro20 and Thr21. Although Arg26 cannot contact the nucleophilic cysteine and has only a marginal influence on pKa,39 prior mutation of Arg26 to Val leads to a particularly marked decrease in enzymatic activities.40 Whether His31 has similar influence on the pKa of Cys27 and/or the enzymatic activities still needs further investigation.