Crystal structure of glutathione‐dependent phospholipid peroxidase Hyr1 from the yeast Saccharomyces cerevisiae (2008)

The accumulation of reactive oxygen species (ROS) in aerobic organisms can cause oxidative stress which results in significant damages to cell constituents. These damages are believed to be related to a couple of degenerative diseases in human beings.1, 2 To eliminate the damages caused by ROS, cells have developed several defense mechanisms.3 As one of the key members during oxidative stress response, the yeast Saccharomyces cerevisiae Hyr1/YIR037W (formerly termed Gpx3) was reported to be a glutathione-dependent phospholipid peroxidase (PhGpx) that specifically detoxifies phospholipid peroxide.4 Moreover, Hyr1 has been identified to sense and transfer the oxidative signal to the transcription factor Yap1.5 Upon accumulation of H2O2, the Hyr1-Cys36 residue is oxidized to a cysteine sulfenic acid (Cys-SOH)6 and then forms a mixed disulfide bond with Yap1-Cys598. This disulfide bond is further resolved into a Yap1 Cys310-Cys598 intra-molecular disulfide bond, leaving a reduced Hyr1-Cys36 ready for the next cycle of signal sensing. When the oxidative stress is released, thioredoxin Trx2 can turn off this signal transduction pathway by reducing both Hyr1 and Yap1. In vivo experiments also suggested that Hyr1 is more likely an oxidative signal sensor and transducer, instead of a hydroperoxides scavenger.5 Compared to mammalian GPxs, Hyr1 has two distinct features. First, Hyr1 lacks an oligomerization loop corresponding to the tetramer interface of mammalian GPxs (except that human GPx4 is a monomeric enzyme lacking the tetrameric interface).7 Second, Hyr1 has a nonaligned second cysteine (Cys82) analogous to the resolving cysteine in the 2-Cys peroxiredoxins.8 Homology modeling suggests that these two features are important determinants to the thioredoxin specificity, which makes Hyr1 a thioredoxin-dependent peroxidase rather than a glutathione-dependent one as it was previously annotated.9 Here we reported the crystal structure of Hyr1 from Saccharomyces cerevisiae and activity assay of its active Cys82. Comprehensive structural analyses revealed that the Cys36 thiol adopts an orientation that favors the formation of Cys36-Cys82 intramolecular disulfide bond. The weak electron density of the Cys82-segment (residues 69–86) suggests a high degree of motion of this region, which might act as a mobile lid to control the recognition of substrates or protein partners. Furthermore, the enzymatic assay of wild-type Hyr1 and Cys82Ser mutant confirmed the importance of Cys82, consistent with previous reports.5 The open reading frame of Hyr1/YIR037W gene from Saccharomyces cerevisiae was cloned into a pET28a-derived vector. This construct adds a hexahistidine (6×His) tag to the N-terminus of the recombinant protein, which was overexpressed in E. coli Rosetta (DE3) strain using 2×YT culture medium (5 g of NaCl, 16 g of bactotrypton, and 10 g of yeast extract per liter). The cells were grown at 37°C up to an A600 nm of 0.6. Expression of recombinant Hyr1 was induced at exponential phase with 0.2 mM isopropyl-β-d-thiogalactoside (IPTG) and cell growth continued for another 20 h at 16°C before harvesting. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM HEPES at pH 7.0, 250 mM NaCl). After 3 min of sonication and centrifugation, the supernatant containing the soluble target protein was collected and loaded to a Ni-NTA column (GE Healthcare) equilibrated with binding buffer (50 mM HEPES pH7.0, 250 mM NaCl). The target protein was eluted with a linear gradient of imidazole from 50 to 500 mM, and further purified by gel filtration in a Superdex 75 column (Amersham Biosciences) equilibrated with 50 mM HEPES pH 7.0 and 250 mM NaCl. The purified protein showed a single band in SDS-PAGE and the integrity was checked by mass spectrometry. The protein sample was concentrated to 1 mg/mL and the solvent-exposed lysines were methylated following the protocol published previously.10 The precipitated protein was removed by centrifugation before purification of the soluble fraction with a desalt column pre-equilibrated in 20 mM Tris-HCl pH 7.0, 50 mM NaCl, and 14 mM β-mercaptoethanol. Fractions containing the target protein were pooled and concentrated to 10 mg/mL. The crystals were grown at 289 K using the hanging drop vapor-diffusion techniques, with the initial condition by mixing 1 μL of the protein sample with equal volume of mother liquor (0.2M ammonium acetate, 0.1M sodium acetate, pH 4.6, 30% polyethylene glycol 4000). Typically, crystals appeared in 10 days. The crystal was transferred to the cryoprotectant of the reservoir solution supplemented with 15% glycerol and flash frozen 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) with a MarRearch 345 image-plate detector at School of Life Sciences, University of Science and Technology of China (USTC, Hefei, China). Data were processed with the Program MOSFLM11 and scaled with SCALA.12 The crystal structure of Hyr1 was solved by the molecular replacement method with the program MOLREP13 using the reduced form of poplar PtGpx5 (PDB code 2P5Q) as the search model. The dataset was severely anisotropic, with diffraction limits of 2.0 Å along the a* and b* directions, but only 2.80 Å along the c* direction. For this reason, the data was truncated along c* at 2.80 Å and scaled with the diffraction anisotropy server (see the Supporting Information table).14 Crystallographic refinement was performed using the program REFMAC5.15 After several rounds of manual rebuilding using the graphics program COOT,16 TLS parameters were analyzed with the TLSMD server and introduced in the refinement.17, 18 The water molecules were then placed into the electron density map, resulting in the final model. The structure was finally refined to 2.02 Å with the Rfactor 22.4% and Rfree 25.7%. The stereochemical quality of the final model was verified using the program PROCHECK.19 The structure factor and coordinate were deposited in the Protein Data Bank under the accession code of 3CMI. The final statistics and refinement parameters were listed in Table I. All figures of protein structure were prepared with PyMol.20 Peroxidase activity was monitored by the spectrometric determination of NADPH consumption at 340 nm following the previous method.5 The reaction was carried out in a buffer containing 100 mM Tris-HCl pH 8.0, 0.3 mM NADPH, and 2.68 μM yeast thioredoxin Trx2 and 0.18 μM yeast thioredoxin reductase Trr1. Purified Hyr1 was added to a 200 μL reaction mix to a final concentration of 1.35 μM, and the reaction was started 30 s later by adding H2O2 to 100 μM. The N-terminal 6×His tag and residues 69–86 including the resolving Cys82 can not be located because of weak electron density, suggesting a high degree of structural flexibility of these regions. The overall structure of Hyr1 adopts the typical thioredoxin-like fold,21 consisting of four β-strands clustered as the central β-sheet which is surrounded by three α-helices [Fig. 1(A)]. The crystal structure indicates that Hyr1 is a monomeric protein, which is consistent with our previous gel filtration result.22 Compared to mammalian tetrameric Gpx structures,23 Hyr1 lacks an oligomeration loop that is located at the C-terminus and an extended N-terminal 310 helix. Besides, the characteristic N-terminal β-hairpin in mammalian Gpxs or poplar Gpx5 is not preserved either.23-25 Hydrophobic residues near Cys36 such as Val32, Tyr42, Pro63, and Phe127 are partially exposed to solvent and form a hollow hydrophobic pocket, which suggests a potential role in the substrate binding. It is worth noticing that these residues are well conserved in the Gpx family, but they are shielded from solvent by helix α2 in mammalian Gpx structures and rPtGpx5 (see below for a detailed discussion). A: Cartoon representation of the overall fold of Hyr1, colored and labeled according to the secondary structures. Cys36 thiol was highlighted as sticks and the interrupted points were colored with purple and connected with a gray dashed line. B, C: Superposition of Hyr1 (red) upon rPtGpx5 (cyan) and oPtGpx5 (gray), respectively. The helix α2 of rPtGpx5 and peroxidatic and resolving cysteine of all structures were labeled. D: Hyr1 peroxidase activity assays. Assays were performed separately for three times in the presence of Trx2, Trr1, NADPH, and H2O2, with Hyr1 (filled squares), with Hyr1-Cys82Ser mutant (open triangles), without Hyr1 as a null control (filled circles). Data are expressed as the decrement of OD340 nm against time in minute. The error bars represent the standard deviations. The side chain of Cys36 is pointing toward the exterior of the protein and is solvent-exposed. Homology modeling and biochemical study of Hyr1 has suggested that Gln70 and Trp125 form hydrogen bonds with the Cys36 thiol in the reduced state and play an essential role in modulating the reactivity towards peroxide.6 However, in our structure the thiol of Cys36 is not hydrogen-bonded to any neighboring residues. The side chain of Trp125 is more than 10 Å away from the thiol of Cys36 and Gln70 is not positioned in the structure due to weak electron density. Compared to the mammalian homologue Gpx4 and reduced form of PtGpx5 (rPtGpx5) with sequence identity ranging from 44% to 50%,24, 25 the structure of Hyr1 undergoes large local conformation changes, especially at the loop between helix α1 and strand β1. Taking rPtGpx5 as the reference structure, the beginning of Hyr1 helix α1 undergoes a partial unwinding and rearrangement so that the main chain of Cys36 shift ∼6.0 Å toward the aligned helix α2 of rPtGpx5, with the thiol of Cys36 flipping over by ∼180° to the opposite and becoming more solvent-exposed [Fig. 1(B)]. The missing Cys82-segment corresponds to the rPtGpx5 helix α2, which is just aligned in the position to shield the hydrophobic pocket of Hyr1 from solvent. Superposition of Hyr1 onto the oxidized form of PtGpx5 (oPtGpx5) structure reveals that the Hyr1 Cys36 has a main chain shift of ∼3.7 Å from the oPtGpx5 Cys42, but the thiols of the two equivalent cysteines adopt the same orientation [Fig. 1(C)]. Moreover, oPtGpx5 helix α2 is completely unwound into less-ordered loop with a relatively higher B-factor,24 whereas in our structure this part shows weak electron density because of the high degree of motion. However, no obvious crystallographic evidence supports the presence of Cys36-Cys82 disulfide bond or Cys-SOH intermediate of Cys36 in Hyr1. Considering the lack of any reducing agent throughout purification and crystallization, the present crystal structure of Hyr1 may represent an intermediate state between the reduced and oxidized states, with the active site residues taking the conformations more favorable of forming Cys36-Cys82 disulfide bond. Taken together, the Cys82-segment of Hyr1 may act as a flexible lid covering the substrate binding site. Upon oxidation, it might be moved to facilitate the formation of intra-molecular disulfide bond, and/or the hydrophobic interaction with its substrate or protein partners, such as hydrophobic part of phospholipid peroxide or Yap1.4, 5 The equivalent parts of mammalian tetrameric Gpxs are not reported to show flexibility partially because they are involved in protein dimerization and thus stabilized.9 However, it remains unknown how the substrates or protein partners specifically interact with Hyr1 and what is the actual driven force of this flexible lid. To confirm whether the recombinant Gpx3 has a functional Cys82, we investigated the catalytic properties of Hyr1 and Cys82Ser mutant. In the presence of thioredoxin system (Trx2-Trr1-NADPH), wild-type Hyr1 could consume H2O2 rapidly, while the Cys82Ser mutant totally abolished the activity, indicating the important role of Cys82 in Trx2 specificity [Fig. 1(D)]. In summary, these results and previous reports5 further evidenced our structural analyses of the mobile active site. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.