Crystal structure of Arabidopsis thaliana peroxiredoxin A C119S mutant

Peroxiredoxin A (PrxA) from Arabidopsis thaliana chloroplasts belongs to the 2-Cys Prx family. The crystal structure of a PrxA C119S mutant at 2.6 Å resolution shows the protein exists as a decamer, which was corroborated in solution. The structure is in the reduced state, suitable for the approach of peroxide.

Abstract

Peroxiredoxins (Prxs), a large family of antioxidant enzymes, are abundant in all living organisms. Peroxiredoxin A (PrxA) from Arabidopsis thaliana belongs to the typical 2-Cys Prx family and is localized in the chloroplast. This article reports the crystal structure of a PrxA C119S mutant refined to 2.6 Å resolution. The protein exists as a decamer both in the crystal structure and in solution. The structure is in the reduced state suitable for the approach of peroxide, though conformational changes are needed for the resolving process.

1. Introduction  

Peroxiredoxins (Prxs) are members of an antioxidant enzyme family that are found ubiquitously in all organisms. Prxs play an important role in regulating the peroxide levels within cells, and about 90% of intracellular peroxides are reduced by Prxs (Winterbourn, 2008 ▸; Adimora et al., 2010 ▸; Perkins et al., 2015 ▸; Hall et al., 2009 ▸). It has been shown that the Prxs from both yeast and human cells can be inactivated by H₂O₂, and the formation of cysteine sulfinic acid in Prxs can reverse the inactivation of Prxs (Woo et al., 2003 ▸). Accumulating evidence has shown that Prxs participate in oxidative stress defence, regulate signaling processes and act in reversing the function of the oxidative protein (Perkins et al., 2015 ▸).

In the model plant Arabidopsis thaliana, Prxs are separated into four types based on their different locations and the number of cysteine residues: 2-Cys Prx, type II Prx, Prx Q and 1-Cys Prx (Dietz, 2011 ▸, Liebthal et al., 2018 ▸). The 2-Cys Prx is located in the chloroplast, the primary organelle of plant redox reactions. Like its homologues from yeast and human cells, 2-Cys Prx also has high peroxidase activity shown by its two characteristic cysteines: the peroxidatic cysteine, which reduces the peroxide, and the resolving cysteine, which subsequently forms a disulfide bond with the oxidized peroxidatic cysteine (Barranco-Medina et al., 2008 ▸; Martí et al., 2009 ▸; Pulido et al., 2010 ▸). A recent study reported that 2-Cys Prx serves as a redox-dependent molecular hub within the chloroplast regulatory network (König et al., 2013 ▸). This redox network is mainly composed of two components, the photoreduced ferredoxin (Fd)-dependent thioredoxin (Trx) reductase, and the NADPH-dependent Trx reductase C (NTRC). These two components are integrated by 2-Cys Prx (Puerto-Galán et al., 2015 ▸; Pérez-Ruiz et al., 2017 ▸; Nikkanen et al., 2017 ▸). NTRC controls the redox balance of 2-Cys Prx to maintain the reducing capacity of chloroplast Trxs. Therefore, 2-Cys Prx is essential for chloroplast function. In addition, 2-Cys Prx is also involved in the regulation of photosynthesis by reacting with a thylakoid-associated atypical thioredoxin-type protein, like ACHT1 or ACHT4 (Dangoor et al., 2012 ▸; Eliyahu et al., 2015 ▸). H₂O₂ is a harmful by-product of photosynthesis. ACHT1 reduces 2-Cys Prx by accepting electrons, then the reduced 2-Cys Prx eliminates the H₂O₂ generated during photosynthesis.

Most of the reported structures of 2-Cys Prxs are from human cells, such as Prx3 (PDB entry 5jcg; Yewdall et al., 2016 ▸), Prx4 (PDB entry 3tkp; Wang et al., 2012 ▸) and Prx5 (PDB entry 2vl3; Smeets et al., 2008 ▸). In plants, the structure of 2-Cys Prx has never been solved. A. thaliana has two 2-Cys Prxs, PrxA and PrxB; they share 85% sequence identity and exert the same biological function. The study of the structure of chloroplast 2-Cys Prx is helpful in revealing the molecular mechanism behind its role in the redox regulatory network and photosynthesis. Here, we report the crystal structure of a PrxA C119S mutant refined to a resolution of 2.6 Å. The mutant was designed to study the catalytic activity of PrxA.

2. Materials and methods  

2.1. Plasmid construction  

The PrxA cDNA fragment corresponding to residues 73−266 (the conserved region of Prxs; Fig. S1 of the supporting information) of AT3G11630 was amplified by PCR from an A. thaliana cDNA library with the forward primer 5′-GGAATTCCATATGCCACTGGTTGGAAACAAGG-3′ (the underlined bases correspond to the NdeI site) and the reverse primer 5′-GATCTCGAGGATGGCGGAAAAGTATTCTTTAGACAG-3′ (the underlined bases correspond to the XhoI site). The PCR product was cloned into the expression vector pET-22b(+) (Novagen) and then transformed into the Escherichia coli BL21(DE3) strain. The cDNA of PrxA was used to generate the mutant by substituting Ser for Cys119 using PCR-mediated site-directed mutagenesis. The oligonucleotides of the mutation were 5′-GACTTCACGTTCGTCTCCCCGACCGAAATCACC-3′ (the forward primer) and 5′-GGTGATTTCGGTCGGGGAGACGAACGTGAAGTC-3′ (the reverse primer), the mutation site is shown in bold. In a similar manner, the C119S mutant was cloned into pET-22b(+) and subsequently transformed into the E. coli BL21(DE3) strain. The recombinant expression plasmids, including the point mutation, were verified by DNA sequencing (Sangon Biotech, Shanghai, China). The expressed recombinant PrxA C119S mutant protein contains a C-terminal His₆ tag (Table 1 ▸).

Table 1. Recombinant protein information.

Source organism	Arabidopsis thaliana
Forward primer (5′ to 3′)	GGAATTCCATATGCCACTGGTTGGAAACAAGG
Reverse primer (5′ to 3′)	GATCTCGAGGATGGCGGAAAAGTATTCTTTAGACAG
Forward primer for mutation	GACTTCACGTTCGTCTCCCCGACCGAAATCACC
Reverse primer for mutation	GGTGATTTCGGTCGGGGAGACGAACGTGAAGTC
Expression vector	pET-22b(+)
Expression host	E. coli BL21(DE3)
Complete amino acid sequence of the construct produced	MPLVGNKAPDFEAEAVFDQEFIKVKLSDYIGKKYVILFFYPLDFTFVSPTEITAFSDRHSEFEKLNTEVLGVSVDSVFSHLAWVQTDRKSGGLGDLNYPLISDVTKSISKSFGVLIHDQGIALRGLFIIDKEGVIQHSTINNLGIGRSVDETMRTLQALQYIQENPDEVCPAGWKPGEKSMKPDPKLSKEYFSAILEHHHHHH

2.2. Overexpression and purification  

To express the recombinant PrxA C119S protein, 20 µl of the preserved strain was used to inoculate a 4 ml LB culture supplemented with 50 µg ml⁻¹ ampicillin at 310 K and then grown overnight. This overnight bacterial culture was inoculated into 1 l LB medium containing 50 µg ml⁻¹ ampicillin and grown to an OD₆₀₀ of 0.8; the culture was then cooled to 289 K, followed by induction with 0.4 mM isopropyl β-d-1-thiogalactopyranoside. Incubation continued for 18 h at 289 K. The cells were harvested by centrifugation at 5000g for 15 min at 277 K.

For the purification of the PrxA C119S protein, the pellets were resuspended in buffer A [20 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM dithiothreitol (DTT), 5%(v/v) glycerol] and disrupted by sonication on ice. Cell debris was removed by centrifugation at 10 000g for 1 h at 277 K. The supernatant of the recombinant protein was loaded onto a Ni-NTA column (Qiagen) pre-equilibrated with buffer A. The column was eluted with 200 mM imidazole supplemented to buffer A. The eluted PrxA protein was pooled and concentrated before loading onto a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with buffer A. Fractions corresponding to PrxA protein were pooled, desalted by dialysis against 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT and 5%(v/v) glycerol, and concentrated to 10 mg ml⁻¹ for crystallization. The presence and purity of the protein were monitored by SDS–PAGE. The protein concentration was determined by a protein Bradford assay (Bio-Rad) with bovine serum albumin as a standard. For determination of the molecular weight, a Superdex 200 10/300 GL column (GE Healthcare) calibrated with a GE high-molecular-weight gel-filtration calibration kit was used.

2.3. Crystallization  

Preliminary screening of the initial crystallization conditions for the PrxA C119S mutant was performed by the sitting-drop vapor-diffusion method using Crystal Screen and Crystal Screen 2 as well as Index (Hampton Research, USA) at 289 K. Sitting drops were prepared by mixing equal volumes (1 µl) of protein solution [15 mg ml⁻¹ in 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 5%(v/v) glycerol] and reservoir solution and were allowed to equilibrate against 100 µl of reservoir solution in 48-well plates. The best crystals were grown in a solution consisting of 0.24 M lithium sulfate, 0.1 M Tris–HCl pH 8.5 and 26%(w/v) PEG 4000 (Table 2 ▸).

Table 2. Crystallization conditions.

Method	Sitting-drop vapor-diffusion
Plate type	48-well plates
Temperature (K)	289
Protein concentration (mg ml−1)	10
Buffer composition of protein solution	20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 5%(v/v) glycerol
Composition of reservoir solution	0.24 M lithium sulfate, 0.1 M Tris–HCl pH 8.5, 26%(w/v) PEG 4000
Volume (µl), ratio of drop	2, 1:1 ratio of protein:reservoir solution
Volume of reservoir (µl)	100

2.4. Data collection and processing  

The diffraction data for a PrxA C119S single crystal were collected at beamline BL17U of the SSRF at a wavelength of 0.9788 Å at 100 K, using a Dectris EIGER 16M detector. The crystals were soaked in reservoir solution with an additional 20%(v/v) glycerol and flash-cooled in liquid nitrogen before data collection. Data were indexed, integrated and scaled using the HKL-2000 package (Otwinowski & Minor, 1997 ▸). Detailed statistics for data collection are shown in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the highest-resolution shell.

Diffraction source	BL17U, SSRF
Wavelength (Å)	0.9788
Temperature (K)	100
Detector	Eiger 16M
Crystal-detector distance (mm)	350
Rotation range per image (°)	1
Total rotation range (°)	200
Exposure time per image (s)	0.5
Space group	C2
Unit-cell parameters
a, b, c (Å)	124.21, 216.19, 98.45
α, β, γ (°)	90, 101.47, 90
Mosaicity (°)	0.8
Resolution (Å)	50.00–2.60 (2.69–2.60)
Total no. of reflections	274204
No. of unique reflections	74467
Completeness (%)	95.9 (94.1)
Redundancy	3.7 (3.4)
〈I/σI〉	9.9 (1.5)†
R meas ‡	0.148 (1.142)
CC1/2	0.995 (0.662)
Overall B factor from Wilson plot (Å2)	39.8

^†Mean I/σI falls below 2.0 in the 2.80–2.69 Å resolution shell.

^‡ R _meas = , where I _i(hkl) and 〈I(hkl)〉 represent the diffraction-intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements.

2.5. Structure solution and refinement  

The structure of the PrxA C119S mutant was solved by molecular replacement with MORDA (Vagin & Lebedev, 2015 ▸) using a monomer of Pseudosciaena crocea Prx4 (PDB entry 3qpm; Lian et al., unpublished work; ∼57% sequence identity) as the search model. The model was rebuilt using COOT (Emsley et al., 2010 ▸) and refined with PHENIX (Adams et al., 2010 ▸). The refinement statistics are shown in Table 4 ▸.

Table 4. Structure refinement.

Values in parentheses are for the highest resolution shell.

Resolution (Å)	50.00-2.60 (2.63-2.60)
Completeness (%)	95.5 (88.9)
σ cutoff	1.360
No. of reflections, working set	70550 (2377)
No. of reflections, test set	3762 (152)
Final R cryst	0.196 (0.302)
Final R free	0.244 (0.329)
No. of non-H atoms
Protein	15320
Water	510
Total	15830
R.m.s. deviations
Bonds (Å)	0.006
Angles (°)	0.814
Average B factors (Å2)
Protein	39.8
Water	39.0
Ramachandran plot
Favored regions (%)	96.9
Additionally allowed (%)	3.1
Outliers (%)	0

3. Results and discussion  

The crystals of the PrxA C119S mutant belonged to space group C2. Native Patterson analysis performed using MOLREP (Winn et al., 2011 ▸) suggested that there was a pseudo-translation along the c direction. The structure was resolved using the molecular replacement method and refined to a resolution of 2.6 Å, with R _cryst and R _free values of 0.196 and 0.244, respectively. There were ten molecules per asymmetric unit, with a Matthews coefficient of 2.84 ų Da⁻¹ (Matthews, 1968 ▸) and a solvent content of 56.7%. Ten molecules existed as two half rings, each of which contained five molecules and formed a donut-shaped decamer with a symmetry-related half ring (Fig. S2), which is consistent with the gel-filtration result (Fig. 1 ▸ a) and the fact that most typical 2-Cys Prxs exist as decamers in the reduced state (Perkins et al., 2015 ▸). The donut has a maximum diameter of ∼130 Å, an inside diameter of ∼60 Å and a thickness of ∼40 Å (Fig. 1 ▸ b). The PrxA decamers are stacked to form tubes in the crystal structure (Fig. S3). It was reported that several typical 2-Cys Prxs can form filaments in a similar manner that can act as molecular chaperons (Gourlay et al., 2003 ▸; Saccoccia et al., 2012 ▸; Radjainia et al., 2015 ▸), but we have no further evidence to conclude that the tubular structures of PrxA in the crystal form are physiologically functional because high-order assemblies of PrxA decamers have not been observed in solution to date.

Figure 1.

Overall structure of PrxA from A. thaliana. (a) Gel filtration result of PrxA C119S, elution volume of molecular weight standards are shown in the box. (b) Decameric structure of PrxA. Two subunits of each dimer are colored cyan and magenta, respectively. The crystallographic twofold axis is indicated. (c) Ribbon diagram of a PrxA monomer, the partner subunit is shown as a gray surface.

There are no significant differences between the structures of each subunit, except for the C-terminal regions of two sub­units, which are involved in crystal packing. The core regions (amino acids 73–252) of each subunit can be superimposed well with root-mean-square deviations (RSMD) of 0.14–0.27 Å. The structure of PrxA falls into the typical 2-Cys peroxiredoxin fold with six α-helices and seven β-strands, which exhibit a typical Trx fold (ββαβαβαβαββαα). Seven β-strands form a β-sheet, with α1 and α4 on the inner side and α2, α3 and α5 on the outer side of the donut, while α6 and a loop stretch out from the core region and interact with a partner subunit (Fig. 1 ▸ c). Three-dimensional structure-based BLAST results generated by the DALI server (Holm & Sander, 1995 ▸) showed that the most similar structure in the PDB is human peroxiredoxin-2 (PDB entry 1qmv, ∼63% sequence identity, Schröder et al., 2000 ▸), with a Z score of 31.4 and an RMSD of 0.7 Å over 197 residues within one subunit.

The basic biological unit of PrxA is a dimer, consistent with other typical 2-Cys peroxiredoxins (Perkins et al., 2015 ▸). The dimeric interface buries ∼3720 Ų (an average of five dimeric interfaces) of surface area. Hydrogen bonds between main-chain atoms at β7 of each subunit, aided by hydrogen bonds between the side-chain atoms of Gln207, His208, Thr210, Asn212 and Asn213 from each β7, assemble two β-sheets from two subunits into a 14-strand β-sheet. (Fig. 2 ▸ b). Hydrogen bonds and hydrophobic interactions between the α5 and the β7–α5 loop of each subunit stabilize the dimeric structure. The C-terminal region, including the α5–α6 loop and α6, interacts with the β7–α5 loop, α2, the β3–α2 loop and the α3–β5 loop of a partner molecule, contributing to about half of the total buried surface area (Fig. 2 ▸ a). The hydrophobic interactions between side chains of α3 and the β3–α2 loop of each dimer build up the biological assembly, a decamer, with a buried surface of ∼1630 Ų (an average of five interfaces) between each adjacent dimer.

Figure 2.

The dimeric structure of PrxA. (a) Ribbon diagram of a PrxA dimer, the peroxidatic and resolving cysteines are shown as dots. (b) Interactions between β7 of each subunit. Hydrogen bonds are illustrated by blue dashed lines. (c) Superimposition of PrxA C119S (cyan and magenta) with fully folded (orange, PDB entry 1qmv; Schroder et al., 2000) and local unfolded (gray, PDB entry 5ijt) human peroxiredoxin-2.

The active site of the PrxA C119S mutant is present in the fully folded conformation (Fig. 2 ▸ c). Ser119, which mimics the reduced state of the peroxidatic Cys119, is located in the N-terminus of α2 and away from the dimeric interface. The hydroxyl group of Ser119 (which represents the thiol group of Cys119) forms a hydrogen bond with the guanidine group of Arg195, which is involved in a hydrogen-bonded network consisting of the carboxyl group of Glu122, the guanidine group of Arg218 and the main-chain carbonyl group of Leu214 (Fig. 3 ▸ a). The hydrogen bond between Cys119 and Arg195 is conserved in all peroxiredoxins and is crucial for their antioxidant enzyme activity (Perkins et al., 2015 ▸). A positively charged cavity surrounded by the guanidine group of Arg195, the hydroxyl group of Thr116 and main-chain amino groups of Phe117, Val118 and Cys119 makes Cys119 suitable for the approach of peroxide (Fig. 3 ▸ b). The thiol group of the resolving Cys241 of the partner subunit is ∼13 Å away from the hydroxyl group of Ser119, making it unsuitable for disulfide-bond formation. Comparing this structure with the peroxiredoxins possessing disulfide bonds, such as human peroxiredoxin-2 in the oxidized state (PDB entry 5ijt; Bolduc et al., unpublished work), suggested that conformational changes of both the peroxidatic cysteine and the resolving cysteine are necessary for the resolving process (Fig. 2 ▸ c).

Figure 3.

The active site of PrxA. (a) The hydrogen-bonded network of Ser119. Hydrogen bonds are illustrated by blue dashed lines. (b) The positively charged substrate-binding cave adjacent to Ser119.

The amino-acid sequence of catalytic sites and the dimeric interface are conserved among typical 2-Cys peroxiredoxins; however, the amino acids on the surface of the decamer are more variable. The evolutionary conservation calculated from 150 homologues by ConSurf (Ashkenazy et al., 2016 ▸) is shown in Fig. 4 ▸. The variable regions of PrxA, including β1, β2, α1, the C-terminal of α2, the α3–β5 loop and α6 may determine its photosynthesis-specific functions.

Figure 4.

Conservation analysis of PrxA in (a) side view, (b) inner view and (c) outer view of the donut-shaped decamer. The surface is colored according to conservation calculated by ConSurf. The partner subunit of the dimer is shown as a yellow ribbon. The peroxide-binding cavity is enclosed in the green dashed circle, and the interface with another dimer in the same decamer is enclosed in the red dashed circle. The conservation color scale is also shown.

4. Conclusions  

We determined the crystal structure of the PrxA C119S mutant, which exists as a decamer assembled from five dimers. Mutation of the peroxidatic cysteine to serine stabilizes the structure in the reduced state which is ready for peroxide reduction, but conformational changes are needed for the subsequent resolving process.

Supplementary Material

PDB reference: Arabidopsis thaliana peroxiredoxin A C119S mutant, 5zte