Crystal structures of human peroxiredoxin 6 in different oxidation states

Biochemical and Biophysical Research Communications xxx (2016) 1e6

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Crystal structures of human peroxiredoxin 6 in different oxidation states Kyung Hee Kim a, b, Weontae Lee b, **, Eunice EunKyeong Kim a, * a b

Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2016 Accepted 24 June 2016 Available online xxx

Peroxiredoxins (Prxs) are a family of antioxidant enzymes found ubiquitously. Prxs function not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Since reactive oxygen species are involved in many cellular metabolic and signaling processes, Prxs play important roles in various diseases. Prxs can be hyperoxidized to the sulfinic acid (eSO2H) or sulfonic acid (eSO3H) forms in the presence of high concentrations of H2O2. It is known that oligomerization of Prx is changed accompanying oxidation states, and linked to the function. Among the six Prxs in mammals, Prx6 is the only 1-Cys Prx. It is found in all organs in humans, unlike some 2-Cys Prxs, and is present in all species from bacteria to humans. In addition, Prx6 has Ca2þ-independent phospholipase A2 (PLA2) activity. Thus far only the crystal structure of Prx in the oxidized state has been reported. In this study, we present the crystal structures of human Prx6 in the reduced (SH) and the sulfinic acid (SO2H) forms. © 2016 Published by Elsevier Inc.

Keywords: Prx6 1-Cys Prx Active site Oxidation Reduction Crystal structure

1. Introduction Peroxiredoxins (Prxs) are a family of antioxidant enzymes found ubiquitously in various cellular compartments, such as the cytosol, nucleus, mitochondria, and endoplasmic reticulum [1]. Based on the location and the number of conserved cysteine residues involved in catalysis they are divided into three classes: typical 2Cys Prxs, atypical 2-Cys Prxs and 1-Cys Prxs. In humans, there are six Prxs (Prx1-6): Prx1-4 and Prx5 are categorized as typical 2-Cys and atypical 2-Cys, respectively, while Prx6 is categorized as the only 1-Cys Prx [2]. They all share the same basic catalytic mechanism, in which a redox-active cysteine (peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the peroxide substrate. However, the recycling of sulfenic acid back to a thiol distinguishes the three classes. The 2-Cys Prxs contain the peroxidatic- and resolving cysteines, which form a disulfide bond to reduce the oxidized form of the peroxidatic cysteine, while the 1-Cys Prxs do not possess the resolving cysteine. In 2-Cys Prxs, thioredoxin is the

Abbreviations: Prx, peroxiredoxin; hPrx, Prx from human; GSH, glutathione; GST, glutathione S-transferase; PLA2, phospholipase A2; MR, molecular replacement. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Lee), [email protected] (E.E. Kim).

reductant while the 1-Cys Prxs are reduced by glutathione in the presence of glutathione S-transferase p [3]. Both 2-Cys Prxs and 1Cys Prxs can be hyperoxidized to the sulfinic acid (eSO2H) or sulfonic acid (eSO3H) forms in the presence of high concentrations of H2O2. However, while the hyperoxidized forms of 2-Cys Prxs can be reversed to restore peroxidase activity by the ATP-dependent enzyme sulfiredoxin, the hyperoxidation of 1-Cys Prxs cannot be restored [4]. Prxs are known to function not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Because reactive oxygen species are involved in many cellular metabolic and signaling processes, Prxs play important roles in various diseases, particularly in inflammation, cancer, and neurodegenerative diseases [5]. Structural studies have shown that all Prxs contain a thioredoxin fold with insertions forming additional secondary-structural elements. The peroxidatic cysteine, in the highly conserved Pro(Xxx)3-Thr/Ser-(Xxx)2-Cys motif present in all Prx classes, is located in a narrow solvent-accessible pocket surrounded by highly conserved residues. While atypical 2-Cys Prx exists as a monomer, all other Prxs, e.g. typical 2-Cys Prxs and 1-Cys Prx, are found as homodimers with the C-terminus of one subunit reaching across the dimer interface to interact with the other subunit. Interestingly, some typical 2-Cys Prxs were found to form toroid-shaped complexes with a pentameric arrangement of dimers [6]. Oligomerization is reported to be redox-sensitive; either the reduced or over-

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oxidized form of the enzyme favors the decameric state [7], and other factors such as post-translational modifications, ionic strength, and pH have been reported to affect the equilibrium as well. For example, in the case of Prx1, phosphorylation at Thr90 was reported to induce the high-molecular weight complex by exposing the hydrophobic region [8], and a recent study showed that conformational changes occur near the peroxidatic cysteine associated with pH-induced decamerization in Prx1 [9]. The oligomeric state of the structure is linked to the chaperone activity [10]. Prx6, the only 1-Cys Prx in mammals, is found in all organs in humans, unlike some 2-Cys Prxs, and is present in all species from bacteria to humans [11]. In addition, Prx6 is unique because it has Ca2þ-independent phospholipase A2 (PLA2) activity; Prx6 can reduce oxidized phospholipids at neutral pH and hydrolyze phospholipids at acidic pH [12]. The phospholipid oxidized by H2O2 binds to Prx6 at cytosolic pH ranges of 7.0e7.4, whereas the reduced phospholipid binds to Prx6 at a maximum of pH 4 [13]. Phospholipase A2 activity is reportedly increased dramatically in hyperoxidation states [14]. Therefore, Prx6 is bifunctional, showing peroxidatic activity involving Cys47 as well as PLA2 activity involving Ser32, His26, and Asp140 [15]. In the case of Prx6, the crystal structure of only the oxidized state was reported previously (PDB code: 1PRX) [16]. Because Prx6 has similar structural features as Prx1, possible oligomerization was suggested for Prx6 [11]. In this study, we present the crystal structures of human Prx6 in the reduced (SH) and the sulfinic acid (SO2H) forms. 2. Materials and methods 2.1. Expression and purification of human Prx6 Human Prx6 cDNA (SwissProt entry P30041) was amplified by PCR using the forward primer 50 -GCCGTGACGGAATTCATGCCCGGAGGTCTGCTTC-30 (EcoRI site underlined) and reverse primer 50 GCTAGGCATCTCGAGTCAAGGCTGGGGTGTGT- AG-30 (XhoI site underlined). The PCR product was cloned into the Escherichia coli expression vector pET28a (Novagen, Madison, WI, USA) containing a hexahistidine tag to the N-terminus. The recombinant vector was transformed into the E. coli BL21 (DE3) expression strain. A single colony of transformants was selected and inoculated in LuriaeBertani medium containing ampicillin (50 mg mL1). The cells were grown at 37  C until the optical density reached OD600 ¼ 0.6 and were then induced with 1 mM isopropyl-b-d-thiogalactopyranoside. Cell growth continued for 20 h at 18  C before harvesting the cells by centrifugation and resuspension in lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 2 mM b-mercaptoethanol). After sonication and centrifugation, the supernatant containing 6X Histagged Prx6 was loaded onto a nickel-nitrilotriacetic acid affinity chromatography (GE Healthcare Life Sciences, Little Chalfont, UK) equilibrated with 50 mM Tris at pH 8.0, 150 mM NaCl, 2 mM bmercaptoethanol. Proteins were further purified by gel filtration chromatography on a Superdex 75 26/60 column (GE Healthcare Life Sciences) equilibrated with 20 mM HEPES at pH 7.0, 2 mM EDTA, and 1 mM 1,4-dithio-DL-threitol. The sulfinic acid form was prepared by adding 10 mM H2O2 to the purified Prx6 followed by final gel filtration with 20 mM HEPES at pH 7.0, 2 mM EDTA and 1 mM 1,4-dithio-DL-threitol. 2.2. MALDI-TOF/MS analysis In order to confirm the oxidation state of the prepared Prx6 samples, we carried out mass analysis by MALDI-TOF/MS (Voyager DE-PRO, Applied Biosystems, Foster City, CA, USA). Both samples were loaded onto an SDS-15% (w/v) polyacrylamide gel and the bands were cut from the gel. The gel slices were washed three times

with 25 mM ammonium bicarbonate and 50% acetonitrile solution for 1 h and dried prior to digestion by trypsin (12.5 ng mL1) (Promega) in 25 mM ammonium bicarbonate for 16 h at 37  C. The peptides from the gel were extracted with 60% acetonitrile solution containing 0.5% trifluoroacetic acid. After evaporation of the solvent, the peptides were dissolved in 0.05% trifluoroacetic acid and 5% acetonitrile solution. One microliter of the peptide mixture was mixed with the same volume of the matrix solution of 5 mg mL1 acyano-4-hydroxy-cinnamic acid solution in 50% acetonitrile containing 0.1% trifluoroacetic acid, and the mass values of peptides were determined. The results are shown in Fig. 1. 2.3. Crystallization, data collection and processing Initial screening for the crystallization condition was performed using a Hydra II Plus One (MATRIX Technology) robotics system and 96-well Intelli plates (Hampton Research, Aliso Viejo, CA, USA) at 295 K, and the hits were further optimized using the hanging drop method. Diffraction-quality crystals for the reduced and sulfinic acid forms were obtained by mixing equal volumes of 22 mg mL1 of Prx6 with a reservoir solution containing 100 mM magnesium formate, 18% (v/v) polyethylene glycol 3350, and 100 mM HEPES at pH 7.5 and 20% (v/v) PEG 8000, respectively. The X-ray diffraction data for the two forms were collected at 100 K in a liquid nitrogen stream using synchrotron radiation (Pohang Light Source, Pohang, Korea). Both crystals belonged to the space group P212121 with six molecules per asymmetric unit and had unit cell dimensions of a ¼ 94.353 Å, b ¼ 106.349 Å, c ¼ 165.504 Å, and a ¼ b ¼ g ¼ 90 for the reduced form and a ¼ 94.419 Å, b ¼ 106.539 Å, c ¼ 167.915 Å, and a ¼ b ¼ g ¼ 90 for the sulfinic acid form. Crystals diffracted to 2.5 Å and 2.9 Å resolution, respectively. The data were processed and scaled using the HKL2000 program suite [17]. The statistics are summarized in Table 1. 2.4. Structure determination and refinement The reduced and sulfinic acid form structures were solved by molecular replacement method using the program MOLREP of the CCP4 suite using Prx 6 from Arenicola marina (PDB code: 2V2G) as a search model. All attempts using human Prx6 (PDB code: 1PRX) as the search model failed to yield a solution. Model building was conducted using the programs Coot and refined with the programs CNS [18], REFMAC [19], and PHENIX [20]. After manual rebuilding, the water molecules were placed into the electron density map, resulting in the final model. The stereochemical quality of the final model was confirmed using the program PROCHECK [21]. Figures were prepared using the program PyMOL [22]. The coordinates have been deposited in the Protein Data Bank (see Table 1). 3. Results and discussion 3.1. Overall structure of Prx6 Crystal structures of human Prx6 in the reduced and sulfinic acid forms were determined and refined to final R-values of 23.5% (Rfree ¼ 29.3%) and 25.3% (Rfree ¼ 27.1%) at 2.5 and 2.9 Å resolution, respectively. Statistics on data collection and refinement are summarized in Table 1. MALDI-TOF analysis showed that the two samples prepared were in two different states with peaks at 1339.5884 and 1371.6680 for the reduced and sulfinic acid form, respectively, as shown in Fig. 1A. The electron density maps calculated using atoms within an 8.0 Å radius and Cys47 omitted show clear density of the reduced and the sulfinic acid form of the catalytic Cys47 (Fig. 1B).

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Fig. 1. Redox states of Prx6. (A) Mass spectrometric analysis of the tryptic fragments of redueced and sulfinic acid acid forms of Prx6 confirm the oxidation states of Cys47. (B) The electron density omit maps contoured at 1.0s level around the peroxidatic Cys47 of both forms.

Table 1 Crystallographic table of reduced and sulfinic acid form. Reduced Data sets Beam Line X-ray wavelength (Å) Resolution range (Å) Space group Unit-cell parameters (Å)

Total/unique reflections Completeness (%) Mean I/s (I) Rmerge (%)b Refinement statistics Resolution range (Å) R/Rfree (%)c No. of protein atoms No. of water molecules Average B-factor (Å2) bond length (Å)/angle (deg.) PDB entry

Sulfinic acid

PAL 4A 1.0000 50.0e2.5 (2.59e2.50)a 50.0e2.9 (3.00e2.90)a P212121 P212121 a ¼ 94.353 a ¼ 94.419 b ¼ 106.449 b ¼ 106.539 c ¼ 165.504 c ¼ 167.915 a, b, g ¼ 90 a, b, g ¼ 90 1,962,787/58,546 3,751,733/38,456 98.9 (97) 97.9 (95.8) 25.2 (2.5) 11.2 (2.2) 7.1 (39.5) 13.3 (35.8) 37.9e2.5 23.5/29.3 10,379 124 50.9 0.004/0.815 5B6M

30.0e2.9 25.3/27.1 10,420 157 49.0 0.021/2.314 5B6N

a

Values in parentheses refer to the highest resolution shell. P P P P Rmerge ¼ h i|I(h,i)-〈I(h)〉|/ h iI(h,i), where I(h,i) is the intensity of the ith. measurement of reflection h, and〈I(h)〉 is the mean value of I(h,i) for all i measurements. c Rfree is calculated from the randomly selected 10% set of reflections not included in the calculation of the R-value.

each monomer, resulting in an extended 10-stranded b-sheet. In addition, hydrophobic residues, e.g. Leu145, Ile147, Leu148, and Tyr149, contribute to further stabilization. Furthermore, the C-terminal domain folds over the N-terminal thioredoxin fold of the other monomer. In particular, Arg155, Thr177, Val179, Asp180, Pro191, Ser213, Tyr217, and Tyr220 of the C-terminal domain make both hydrophobic and polar interactions with Phe43, Pro45, Thr48, Thr49, Trp82, Asp85, and Tyr89 of the N-terminal domain. The total dimer interfaces range from 2100 to 2400 Å2, which correspond to 18e21% of the accessible surface area for each monomer. The interface at the b-sheet is approximately 1100 Å2, which corresponds to approximately 12% of the accessible surface area for the N-terminal thioredoxin fold, while the corresponding value at the N- and C-terminal domains is approximately 750 Å2. The dimer interface reported for the sulfenic acid structure are similar to the values observed here [16]. The root mean square deviation (RMSD) for the six molecules range from 0.3 to 1.0 Å for the 199 to 221 Ca. And the RMSD between the reduced and sulfinic acid forms is 0.6 Å for 1311 Ca atoms.

3.2. Peroxdatic active sites of Prx6 in different redox states

b

In both crystal forms, there are six molecules (AeF) in the asymmetric unit (Fig. 2). However, they do not form a quaternary structure, but form a tight homo-dimer related by a noncrystallographic two folds. The two molecules in the dimer are in an antiparallel arrangement as seen in the figure. Each monomer has two domains: the N-terminal domain in the thioredoxin fold with five a-helices and seven b-strands followed by the smaller Cterminal domain with one a-helix and three b-strands. The two domains in the monomer showed minimal interaction. The dimer creates a hydrogen bonding network between the b7 strands of

The catalytically active Cys47 located at the N-terminal end of the a2 helix is at the narrow base pocket formed between the thioredoxin fold and the C-terminal domain of the second molecule in the dimer; the two peroxidative cysteines are ~34 Å apart (Fig. 2). The peroxidatic cysteine is part of the highly conserve motif of ‘40Pro-Arg-Asp-Phe-Thr-Pro-Val-Cys-Thr-Thr-50Glu’, with the residues in bold absolutely conserved across species (Fig. 3A). Two prolines in this motif shield the active site, while the backbone atoms Asp42 and Phe43 lie above the indole ring of Trp82, which is also highly conserved (Fig. 3B). The side chains of Glu50 form hydrogen bonds with the guanidinium moiety of highly conserved Arg132, which is thought to be involved in catalysis (Fig. 3B), and Arg155, which is below Arg132. The guanidinium moiety of Arg132 further interacts with the Sg of Cys47 in both the reduced and sulfinic acid forms as shown in the figure. The interactions seen

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Fig. 2. Overall structure of Prx6. Arrangement of six molecules in the asymmetric unit are shown on the left with each chain labelled. Homodimer formed by chains A and B are shown on the right with the peroxidatic and PLA2 active sites indicated by red circles on chain A.

here are the same as those observed in the sulfenic acid structure [16]. In the sulfinic acid form, Thr44 stabilizes the hyperoxidized Cys47 (Fig. 3C). However, in the reduced and sulfinic acid forms, His39 hydrogen bonds with Asp42 but interacts with Cys47 in the sulfenic acid form (Fig. 3C). In the sulfenic acid form, Sg of Cys47 is

stabilized by Nε1 of His39, and this reorientation of the imidazole ring of His39 causes in Arg132 to swing away from Cys47 and interact with Glu50 [16]. The structural difference in the conformation of His39 suggests that there is enough room at the peroxidatic active site of Prx6. Fig. 3C shows the key residues involved in

Fig. 3. Peroxdatic active site of Prx6 in reduced, sulfenic acid and sulfinic acid forms. (A) Sequence of Prx6 from human, Arenicola marina, Plasmodium yoelii and Aeropyrum pernix at the peroxidatic sites are aligned using ESPript [29]. Residues in the red and blue boxes are strictly conserved or relatively conserved, respectively. Secondary structure depicted above are of this study with the a-helices and the b-strands indicated by coils and arrows, respectively. (B) Structures of Prx6 in the reduced (lime), sulfenic acid (dark red) and sulfinic acid (cyan) forms are superposed. (C) Peroxidatic catalytic cycle of hPrx6. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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catalysis in the three structures. AphE from Mycobacterium tuberculosis is 1-Cys Prx, whose structures are known for the reduced and sulfenic acid forms [23]. Although they are found to form a tetramer and octamer, respectively, there is practically no structural change between the two oxidation states. In the case of Prx4, a typical 2-Cys Prx, the structures of three different oxidation states were reported [24]. In this case, the key residues at the active sites are almost same in the reduced, sulfenic acid and sulfonic acid forms of Prx4, however, the orientation of the peroxidatic Cys87 differ from one another depending of the oxidation state [24]. 3.3. Phospholipase A2 activity site The lipase motif (GXSXG), which are essential residues in serinebased lipases, is present in Prx6 (residues 30e34), and the transition state analog 1-hexadecyl-3-trifluoroethyl-sn-glycero-2phosphomethanol (MJ33) competitively inhibits the PLA2 activity of Prx6 [25]. Ser32, His26, and Asp140 of Prx6 are thought to be involved in PLA2 activity with Ser32 being involved in both the binding and hydrolysis of the liposome, while His26 is involved only in binding and Asp140 in catalysis [15]. These residues are located on the opposite side of the peroxidatic active site as seen in Fig. 2. The distance between His26 and Asp140, however, is significantly longer in Prx6 (~9.7 Å) compared to that of lysosomal phospholipase A2 (5.0 Å) in all six crystallographically independent molecules of both forms evaluated in this study; in fact, the distance is even longer in the oxidized form, requiring structural rearrangement from what that observed in these crystal forms for PLA2 activity. Interestingly, the phosphorylation of Thr177 is reported to enhance the PLA2 activity of Prx6 [26]. It is possible that phosphorylation of Thr177, which is located at the C-terminal domain, may confer the necessary structural change. It was previously suggested that the phospholipid binds to the monomeric Prx6 with the phospholipid head positioned toward the PLA2 activity site and the acyl chain towards the peroxidatic active site [12]. However, our structural evidence suggests that the dimeric interface of Prx6 is rather large, and it is unlikely that Prx6 becomes a monomer; the phospholipid may bind to the flat surface across the PLA2 active site and peroxidatic active site of the other monomer. The distance between Cys47 and Ser32 is approximately 28 Å and there are a number of positively charged residues forming a groove (Fig. 4A). These results must be confirmed in further studies.

Fig. 4. Structural comparisons of Prx6 in reduced and sulfenic acid forms. (A) Electrostatic surface of Prx6 in the sulfenic acid form. The active sites of peroxidatic and PLA2 sites are indicated by yellow dotted circle. (B) Comparison of Prx6 in the reduced, sulfenic acid and sulfinic acid forms with the connecting loop between a4 and b6 highlighted in lime, dark red and cyan, respectively. This view is rotated by 90 from Fig. 2 around the horizontal axis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Connecting loop between a4 and b6 When the structures of this study were compared to that of the oxidized form, the most striking difference was observed for residues Leu114-Arg132, the loop connecting a4 and b6 (Fig. 4B). As seen in the figure, this loop is packed towards the b3-a2 loop in the reduced structure, while it is packed against the a4 helix in the sulfenic form, with the Ca for residues 119e122 differing by more than 10 Å. The conformation of this loop is approximately the same in all six molecules in both the reduced and sulfinic acid forms and there are no crystal contacts within 10 Å from this loop in all cases, suggesting that the observed conformation does not originate from the crystal packing. However, the average temperature B-value for this loop is significantly higher than that of the rest of the molecule (~32 Å2 vs. ~23 Å2), suggesting that this loop is flexible. In the sulfenic acid form, residues 122e126 are disordered in the second molecule. This loop was previously suggested to be part of the accommodation site for the extended substrate in 1-Cys Prx AhpE from M. tuberculosis [23]. As described above, glutathione (GSH) is the physiological reductant for Prx6. Once Prx6 associates with glutathione S-transferase p (pGST), GSH removes pGST from the

complex, and glutathionylated Prx6 is regenerated to the active form by a second GSH [3,27]. Interestingly, a study using fluorescence titration with Prx6 and pGST fragments found that Pro40Cys47 and Leu148-Phe157 of Prx6 interact with pGST [28]. Based on the close proximity to the peroxidatic cysteine and flexibility of this loop, this loop may be involved in the interaction with pGST to allow easy access for GSH to enter the active site. Conflict of interest The authors have no financial conflicts of interest. Acknowledgements We thank staff at 4A beamline, Pohang Accelerator Laboratory, Korea. This work was supported by grant from the Global Research Laboratory (GRL) program of the Ministry of Science, ICT and Future Planning of Korea (MISP: grant No. NRF 20110021713), and grant from Korea Institute of Science and Technology (2E26360).

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Please cite this article in press as: K.H. Kim, et al., Crystal structures of human peroxiredoxin 6 in different oxidation states, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.125