High-resolution crystal structure of the reduced Grx1 from Saccharomyces cerevisiae

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates. Here, the crystal structure of reduced yGrx1 at 1.22 Å resolution is reported and compared with the existing structures of the oxidized and glutathionylated forms. This revealed structural differences in the conformations of residues neighbouring the Cys27–Cys30 active site, which accompany alterations in the redox status of the protein.

Abstract

Grx1, a cytosolic thiol–disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P2₁2₁2₁ and its structure solution and refinement to 1.22 Å resolution are reported. To study the structure–function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27–Cys30 active site which accompany alterations in the redox status of the protein.

1. Introduction  

Glutaredoxins (Grxs) are intracellular GSH-dependent oxidoreductase enzymes (EC 1.8.1.7). They catalyze reversible thiol–disulfide exchange reactions between protein thiols and GSSG/GSH and hence play a critical role in the maintenance of cellular redox homeostasis (Holmgren, 1989 ▸; Prinz et al., 1997 ▸). Glutaredoxin enzymes act to protect cells from damage caused by reactive oxygen species (ROS) by catalyzing the reduction of protein disulfides (P-SS) and the deglutathion­ylation of mixed disulfides (P-SSG), and the overall direction of the reactions depend on the reduction potential of the GSSG/2GSH couple, the nature of the protein thiols involved and other solution conditions such as the presence of metal ions. In the reduction of glutathionylated disulfides (degluta­thionylation reaction mechanism), the N-terminal active-site cysteine of Grx exists as a thiolate anion and attacks the glutathionyl sulfur of the P-SSG mixed disulfide, forming a Grx enzyme intermediate [Grx(SH)(SSG)] and releasing the reduced protein (P-SH). The Grx mixed disulfide is reduced by GSH, forming oxidized GSSG, which is then reduced to GSH by glutathione reductase and NADPH (Begas et al., 2017 ▸; Ukuwela et al., 2017 ▸, 2018 ▸). Structurally, Grxs share a common structural fold with the thioredoxin (TRX) superfamily, which is represented by a central core of four β-strands surrounded by five α-helices and an active-site CXXC sequence motif (Martin, 1995 ▸; Cave et al., 2001 ▸).

Five Grxs have been characterized from the yeast S. cerevisiae to date. These include Grx1 (yGrx1) and Grx2, which contain an active-site CPYC (Luikenhuis et al., 1998 ▸) motif which participates in redox processes through the formation of a disulfide bond between the two cysteine residues or via glutathionylation of the first cysteine residue (Holmgren, 1989 ▸; Prinz et al., 1997 ▸; Ritz & Beckwith, 2001 ▸). However, other Grxs (Grx3, Grx4 and Grx5) from this organism contain only a single cysteine residue in an active-site CGFS motif, which catalyses the (de)glutathionylation of protein thiol groups using glutathione substrates (reduced, GSH, and oxidized, GSSG; Rodríguez-Manzaneque et al., 1999 ▸). Crystal structures of the yGrx1 enzyme in the oxidized, glutathionyl­ated (Yu et al., 2008 ▸) and glutathionylated C30S mutant (Håkansson & Winther, 2007 ▸) forms have been reported previously (PDB entries 3c1r, 3c1s and 2jac, respectively). Here, we augment these structures with the first structure of yGrx1 in the reduced form in the absence of glutathionylation. A comparison of the structure of reduced yGrx1 (^redyGrx1; PDB entry 6mws, this work) with the structures of the oxidized (^oxyGrx1; PDB entry 3c1r) and glutathionylated (^gluyGrx1; PDB entry 3c1s) forms reveals conformational changes which accompany alterations in the redox status of this protein.

2. Materials and methods  

2.1. Macromolecule production  

The pGEX-6p-1-yGrx1 (GenScript) plasmid was transformed into Escherichia coli strain BL21 CodonPlus (DE3). Cultures were grown at 310 K in Luria Broth (LB) supplemented with ampicillin (100 µg ml⁻¹) and chloramphenicol (35 µg ml⁻¹) to an OD₆₀₀ of 0.8, induced with isopropyl β-d-1-thiogalactopyranoside (IPTG; 0.5 mM) and harvested after 16 h (with shaking) at 298 K.

GST-yGrx1 was purified by GSH affinity chromatography. Frozen cell pellets were thawed at room temperature and resuspended in cell-lysis buffer [phosphate-buffered saline (PBS) pH 7.4, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP)]. The cells were disrupted by passage through a TS series bench-top cell disruptor (Constant Systems) at 241 MPa. Cell debris was removed by centrifugation (Beckman JLA-25.50, 30 000g, 20 min, 277 K) and the soluble fraction was incubated for 2 h at 277 K with glutathione Sepharose 4B resin (GE Healthcare) equilibrated with lysis buffer (PBS containing 1 mM TCEP). The GST tag was cleaved overnight in lysis buffer with PreScission Protease followed by size-exclusion chromatography (SEC; HiLoad 16/600 Superdex 75 pg, GE Healthcare; 20 mM Tris–MES pH 8.0, 150 mM NaCl, 1 mM TCEP). Cleavage of the N-terminal GST tag introduced five additional residues (GPLGS) to the N-terminus of yGrx1 (Table 1 ▸). The purified protein was concentrated to 20 mg ml⁻¹ before storage at 193 K.

Table 1. Macromolecule-production information.

Source organism	S. cerevisiae
DNA source	S. cerevisiae
Expression vector	pGEX-6p-1
Expression host	E. coli strain BL21 CodonPlus (DE3)
Complete amino-acid sequence of the construct	GPLGSMVSQETIKHVKDLIAENEIFVASKTYCPYCHAALNTLFEKLKVPRSKVLVLQLNDMKEGADIQAALYEINGQRTVPNIYINGKHIGGNDDLQELRETGELEELLEPILAN

2.2. Crystallization  

Crystallization trials were conducted using commercially available screens (SaltRx HT and Index HT from Hampton Research) by sitting-drop vapour diffusion in 96-well plates (Molecular Dimensions) using pure yGrx1 samples at two different protein concentrations (10 and 20 mg ml⁻¹). Crystallization drops consisting of equal volumes (0.2 µl) of reservoir and protein solutions were dispensed using a Crystal Gryphon liquid-handling system (Art Robbins Instruments) and were equilibrated against a reservoir of screen solution (50 µl). Plates were incubated at 293 K. Multiple tiny crystals were observed within ten days in conditions F7 and G2 of the Index HT screen [0.2 M ammonium sulfate, 0.1 M bis-Tris pH 6.5, 25%(w/v) PEG 3350 and 0.2 M lithium sulfate monohydrate, 0.1 M bis-Tris pH 5.5, 25%(w/v) PEG 3350, respectively]. Optimization of these conditions was carried out by hanging-drop vapour diffusion in 24-well VDX plates (Hampton Research). Diffraction-quality crystals of yGrx1 grew after 20 days in drops consisting of equal volumes (1 µl) of yGrx1 (16 mg ml⁻¹ in 20 mM Tris–MES pH 8.0, 150 mM NaCl, 1 mM TCEP) and reservoir [0.23 M lithium sulfate monohydrate, 0.1 M bis-Tris pH 5.8, 26%(w/v) PEG 3350] solutions equilibrated against 500 µl reservoir solution. Crystals were cryoprotected in reservoir solution containing 25%(w/v) glycerol before flash-cooling them in liquid nitrogen. Crystallization information is summarized in Table 2 ▸.

Table 2. Crystallization.

Method	Hanging-drop vapour diffusion
Plate type	VDXm plates
Temperature (K)	293
Protein concentration (mg ml−1)	16
Buffer composition of protein solution	20 mM Tris–MES pH 8.0, 150 mM NaCl, 1 mM TCEP
Composition of reservoir solution	0.23 M lithium sulfate monohydrate, 0.1 M bis-Tris pH 5.8, 26%(w/v) PEG 3350
Volume and ratio of drop	2 µl, 1:1
Volume of reservoir (µl)	500

2.3. Data collection and processing  

Diffraction data were recorded on beamline MX2 at the Australian Synchrotron at a wavelength of 0.954 Å at 100 K using an EIGER X 16M detector and were processed with XDS (Kabsch, 2010 ▸) and merged and scaled with AIMLESS (Evans & Murshudov, 2013 ▸). Data-collection statistics are detailed in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	MX2, Australian Synchrotron
Wavelength (Å)	0.953654
Temperature (K)	100
Detector	EIGER X 16M
Crystal-to-detector distance (mm)	150
Total rotation range (°)	180
Space group	P212121
a, b, c (Å)	41.4, 51.8, 57.2
α, β, γ (°)	90.0, 90.0, 90.0
Mosaicity (°)	0.04
Resolution range (Å)	41.36–1.22 (1.25–1.22)
Total No. of reflections	474455 (17110)
No. of unique reflections	37323 (1777)
Completeness (%)	99.9 (98.3)
Multiplicity	12.7 (9.6)
〈I/σ(I)〉	19.4 (4.0)
CC1/2	0.999 (0.969)
R merge	0.053 (0.319)
Overall B factor from Wilson plot (Å2)	14.2

2.4. Structure solution and refinement  

The crystal structure of yGrx1 was solved by molecular replacement using Phaser (McCoy et al., 2007 ▸) from the CCP4 suite (Winn et al., 2011 ▸). The crystal structure of oxidized yGrx1 (Yu et al., 2008 ▸) was used as a search model after the removal of all water molecules. The model was refined using REFMAC5 (Murshudov et al., 2011 ▸) and manual model building was carried out in Coot (Emsley et al., 2010 ▸). Automated water picking was carried out using ARP/wARP (Langer et al., 2008 ▸) and was then checked manually in Coot (Emsley et al., 2010 ▸). The quality of the structure was determined by MolProbity (Chen et al., 2010 ▸). Refinement statistics are summarized in Table 4 ▸.

Table 4. Structure solution and refinement.

Values in parentheses are for the outer shell.

Resolution range (Å)	38.38–1.22 (1.25–1.22)
Completeness (%)	99.9 (98.3)
σ Cutoff	None
No. of reflections, working set	35443
No. of reflections, test set	1820
Final R cryst	0.148 (0.149)
Final R free	0.171 (0.159)
No. of non-H atoms
Protein	854
Sulfate	5
Water	122
Total	981
R.m.s. deviations
Bonds (Å)	0.010
Angles (°)	1.430
Average B factors (Å2)
Protein	13.731
Sulfate	14.822
Water	23.273
Ramachandran plot
Most favoured (%)	99.1
Allowed (%)	100
MolProbity score	0.82
PDB code	6mws

3. Results and discussion  

The ^redyGrx1 crystallized in space group P2₁2₁2₁, with unit-cell parameters a = 41.4, b = 51.8, c = 57.2 Å. The structure was determined by molecular replacement and refined to 1.22 Å resolution. The refinement converged with residuals R _cryst = 14.8% and R _free = 17.1%. The solvent content of the crystal was 60% and the structure shows a single molecule of ^redyGrx1 (residues Val2–Ala109) in the asymmetric unit. Owing to an absence of interpretable electron density for the five additional N-terminal residues (GPLGS) and the N-terminal and C-terminal residues of yGrx1 (Met1 and Asn110, respectively), these were omitted from the final model. The overall fold of ^redyGrx1 (Fig. 1 ▸ a) is similar to those of the previously reported yGrx1 structures (PDB entries 3c1r, 3c1s and 2jac; Yu et al., 2008 ▸; Håkansson & Winther, 2007 ▸), with four mixed β-strands in a central core structure surrounded by five α-helices.

Figure 1.

Cartoon representation of the overall structure of ^redyGrx1. (a) Secondary structures are represented as cartoons, with α-helices and β-strands coloured cyan and pink, respectively. The Cys27 and Cys30 residues are shown as yellow sticks. (b) F _o − F _c difference Fourier electron-density map calculated after omission of the Cys27 and Cys30 residues from the model coordinates. C, O, N and S atoms are coloured cyan, red, blue and yellow, respectively. The F _o − F _c map is contoured at 4σ.

The positions of the side chains (and in particular the thiol groups) of cysteine residues Cys27 and Cys30 were confirmed by the calculation of difference Fourier electron-density maps (using a model with the side chains of these residues removed; Fig. 1 ▸ b). These residues were modelled with a distance between the S atoms of Cys27 and Cys30 of 3.2 Å, which is significantly greater than that observed (2.05 Å) for the ^oxyGrx1 structure (PDB entry 3c1r; Yu et al., 2008 ▸). This indicates that the yGrx1 structure reported here is indeed that of ^redyGrx1 (PDB entry 6mws).

Superposition of the ^redyGrx1 structure with that of ^oxyGrx1 (PDB entry 3c1r; Yu et al., 2008 ▸) gave a root-mean-square deviation (r.m.s.d.) of 0.48 Å for 108 common C^α positions, demonstrating that minimal conformational changes occur to the overall yGrx1 structure on oxidation and/or reduction. However, the reduction of the Cys27–Cys30 disulfide bond and separation of the thiol groups of these residues accompanies conformational rearrangements in the neighbouring protein structure, specifically residues Thr25, Tyr26 and His31 (Fig. 2 ▸). In the ^oxyGrx1 structure the side chain of Tyr26 faces ‘away’ from the Cys27–Cys30 site. In ^redyGrx1 Tyr26 shows a conformation rotated by approximately 180° from that observed for ^oxyGrx1, with the side chain orientated ‘towards’ the Cys27–Cys30 site. This is accompanied by a flip in the orientation of the carbonyl group of residue Thr25, so that in the ^redyGrx1 structure this group participates in a hydrogen-bonding interaction with the side chain of His31, which is also reorientated (Fig. 2 ▸). Interestingly, similar conformations for residues Thr25, Tyr26 and His31 were observed in the structure of ^gluyGrx1 (PDB entry 3c1s; Yu et al., 2008 ▸), which also lacks a disulfide bond between residues Cys27 and Cys30 owing to the glutathionylation of Cys27. In addition, conformational changes of neighbouring amino acids have been reported for E. coli Grx1 (PDB entries 1ego and 1grx; Xia et al., 1992 ▸) by NMR, in which reduction of the active-site Cys11–Cys14 disulfide bond was reported to result in an enhanced rate of exchange for the neighbouring residues Phe6, Gly7 and Ala17, indicating conformational changes in these residues.

Figure 2.

Details of the yGrx1 active site (CPYC) upon reduction (cyan), oxidization (salmon) and glutathionylation (green). Comparison of the ^redyGrx1 (cyan), ^oxyGrx1 (PDB entry 3c1r; salmon) and ^gluyGrx1 (PDB entry 3c1s; green) structures shows that upon the separation of the Cys27 and Cys30 residues owing to reduction or glutathionylation, the Thr25, Tyr26 and His31 residues (shown in stick representation) undergo conformational changes that accompany the change in redox state of the enzyme.

In summary, although ^redyGrx1 crystallized in a different condition and space group to the reported ^oxyGrx1 and ^gluyGrx1 structures, the conformational changes of residues neighbouring the active site (CPYC), particularly Thr25, Tyr26 and His31, is consistent among the three structures. This indicates these changes are not owing to crystal packing and instead reflect the redox state of yGrx1. The precise role that these residues play in the activity of yGrx1 remains to be explored.

Supplementary Material

PDB reference: reduced Grx1, 6mws

Funding Statement

This work was funded by Australian Research Council grant DP140102746 to Megan J. Maher.