Archives of Biochemistry and Biophysics 560 (2014) 10–19

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Structural and biochemical analysis of a type II free methionine-Rsulfoxide reductase from Thermoplasma acidophilum Hyun Sook Kim a,1, Geun-Hee Kwak b,1, Kitaik Lee a, Chang-Hwa Jo a, Kwang Yeon Hwang a,⇑, Hwa-Young Kim b,⇑ a b

Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea

a r t i c l e

i n f o

Article history: Received 7 May 2014 and in revised form 27 June 2014 Available online 17 July 2014 Keywords: Methionine sulfoxide reductase fRMsr Catalysis ROS Resolving Cys Thermoplasma

a b s t r a c t Free methionine-R-sulfoxide reductase (fRMsr) enzymes only reduce the free form of methionine-R-sulfoxide and can be grouped into two types with respect to the number of conserved Cys residues in the active sites. In this work, the crystal structures of type II fRMsr from Thermoplasma acidophilum (TafRMsr), which contains two conserved Cys, have been determined in native form and in a complex with the substrate. The overall structure of TafRMsr consists of a central b-sheet encompassed by a two-a-helix bundle flanking on one side and one small a-helix on the other side. Based on biochemical and growth complementation assays, Cys84 is demonstrated to be the catalytic Cys. The data also show that TafRMsr functions as an antioxidant protein. Structural analyses reveal insights into substrate recognition and orientation, conformational changes in the active site during substrate binding, and the role of active site residues in substrate binding. A model for the catalytic mechanism of type II TafRMsr is suggested, in which intramolecular disulfide bond formation is not involved. In addition, the biochemical, enzymatic, and structural properties of type II TafRMsr are compared with those of type I enzymes. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Reactive oxygen species (ROS),2 generated as by-products of cellular metabolism in aerobic organisms, oxidize various cellular components. The ROS-mediated oxidation of macromolecules (such as DNA, proteins, and lipids) may lead to the development of diseases and accelerate the aging process. The sulfur atom of methionine is susceptible to oxidation by ROS. Methionine oxidation produces two diastereomers, methionine-S-sulfoxide (Met-S-O) and methionine-R-sulfoxide (Met-R-O) [1], reversible through a reductase system.

⇑ Corresponding authors. Address: Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk, Seoul 136-701, Republic of Korea. Fax: +82 2 923 3229 (K.Y. Hwang). Address: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchungno, Namgu, Daegu 705-717, Republic of Korea. Fax: +82 53 620 4341 (H.-Y. Kim). E-mail addresses: [email protected] (K.Y. Hwang), [email protected] (H.-Y. Kim). 1 These authors contributed equally to this work. 2 Abbreviations used: ROS, reactive oxygen species; Met-S-O, methionine-S-sulfoxide; Met-R-O, methionine-R-sulfoxide; Msrs, methionine sulfoxide reductases; DTNB, 5,50 -dithiobis(2-nitro)benzoate; SDS, sodium dodecyl sulfate. http://dx.doi.org/10.1016/j.abb.2014.07.009 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

Methionine sulfoxide reductases (Msrs) are the enzymes responsible for the reduction of Met-O and they play a pivotal defensive role against oxidative stress [2–5]. There are three Msr families, MsrA, MsrB, and fRMsr, with distinct substrate specificity [6–8]. MsrA is specific for Met-S-O [9] and has significant reduction activity toward free Met-S-O as well as peptidyl Met-S-O [10]. MsrB catalyzes the stereospecific reduction of Met-R-O [11] but is mainly active on peptidyl Met-R-O, with a very low activity toward free Met-R-O [10]. The fRMsr enzymes, the most recently identified Msr enzymes, only reduce the free form of Met-R-O [12] and cannot act on peptidyl Met-R-O. Interestingly, the fRMsr enzymes contain a GAF domain, a ubiquitous motif in cyclic GMP phosphodiesterases [12]. The fRMsr enzymes only exist in unicellular organisms, including Escherichia coli, and not multicellular organisms [13]. There are two variants of fRMsr proteins, classified by the number of conserved Cys residues [13]. Type I fRMsrs, such as the enzymes from E. coli and Staphylococcus aureus, possess three conserved Cys residues (Cys50, Cys60, and Cys84; numbering based on Thermoplasma acidophilum fRMsr), whereas type II fRMsrs contain two conserved Cys residues (Cys60 and Cys84). The structures and catalytic mechanism of fRMsrs have only been characterized in type I enzymes [14,15]. The catalytic

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mechanism of type I fRMsr involves a sulfenic acid chemistry [14,15], similar to the mechanisms of MsrA and MsrB, in which Cys84 functions as a catalytic residue (Scheme 1). After its attack on the sulfoxide moiety of the substrate, the catalytic Cys84 is converted into a sulfenic acid intermediate with the concomitant release of the product Met. Cys50 acts as a resolving residue and interacts with the Cys84 sulfenic acid, forming an intramolecular disulfide bond. The disulfide Cys84–Cys50 bond is then reduced by reductants. Thioredoxin (Trx) is considered the in vivo reductant, while dithiothreitol (DTT) is used as the in vitro reductant. However, the structure and catalytic mechanism of type II fRMsr enzymes are unknown. In this study, we determined the crystal structures of a type II fRMsr from T. acidophilum (TafRMsr), including the native and substrate-bound forms. Biochemical and kinetic analyses of wild type TafRMsr and mutants, in which Cys residues were replaced with Ser, were performed. A catalytic mechanism of TafRMsr is proposed, in which no intramolecular disulfide bond formation is involved. In addition, differences in the structures and catalytic mechanisms between type I and type II fRMsr enzymes are discussed.

Materials and methods Cloning and purification of TafRMsr The coding region of the TafRMsr was PCR-amplified using its genomic DNA and cloned into NdeI/XhoI sites of pET21b (Novagen). The resulting construct, named pET-TAfRMsr, encoded the full-length TafRMsr with a C-terminal His-tag (LEHHHHHH). C15S, C60S, and C84S mutants, in which Cys is replaced with Ser, were generated by site-directed mutagenesis using the construct pET-TAfRMsr. All constructs were verified by DNA sequencing. The expression plasmid was introduced into E. coli BL21(DE3) cells. After growing the cells in LB medium containing 100 lg/ml ampicillin at 37 °C to an OD600 of 0.8, protein overexpression was induced overnight by adding 0.5 mM IPTG at 18 °C. The cells were then pelleted, resuspended in an extraction buffer (30 mM Tris– HCl, pH 8.0, 10% glycerol, and 40 mM imidazole), and sonicated for disruption. After centrifugation at 17,000g for 20 min, the supernatant of the lysate was loaded onto a HiTrapTM column (GE Healthcare) and washed with the extraction buffer. The protein was eluted by a linear gradient of imidazole (40–500 mM). The TafRMsr protein was further purified by size-exclusion

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chromatography on a HiLoad 26/60 SuperdexTM 200 (Amersham Biosciences). Yeast constructs and growth complementation assay Wild type and mutants of TafRMsr with a C-terminal His tag were PCR-amplified using the pET-based constructs and cloned into BamHI/SalI sites of p425 GPD yeast vector. All constructs were verified by DNA sequencing. The yeast expression constructs were introduced into Saccharomyces cerevisiae cells deficient in triple msrA/msrB/fRmsr genes (MATa his3 leu2 met15 ura3 DmsrA::URA3 DmsrB::KAN DfRmsr::HIS3) [13] using the lithium acetate method. Transformants were selected for leucine prototrophy. Growth complementation assays for the cells containing TafRMsr constructs were performed on a yeast nitrogen base minimal medium supplemented with 2% glucose (YNBD) agar medium in the presence of 0.14 mM Met or 0.28 mM Met-(R,S)-O, as previously described [16]. At least two biological replicates were done. Antioxidant assay The triple msrA/msrB/fRmsr-deleted S. cerevisiae cells containing the p425 vector alone or p425-based TafRMsr constructs were grown aerobically at 30 °C in YNBD media. The overnight cultures were each adjusted to an OD600 of 2.5, 0.25, and 0.025 via serial dilution. Each diluted sample (5 ll) was spotted onto YNBD agar medium in the presence of 0.3 mM H2O2. The spotted cells were incubated for 2 days at 30 °C and cell growth was monitored. Activity and kinetic analysis of TafRMsr The reaction mixture (200 ll) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 0.2 mM NADPH, 0.1 mM EDTA, 10 lg E. coli Trx, 14 lg human Trx reductase 1, 0.05–1.6 mM free Met-R-O, and 3 lg TafRMsr. The reactions were carried out at 25 °C for 5 min, and a decrease in the absorbance of NADPH was monitored at 340 nm using a spectrophotometer UV-160A (Shimadzu). A control for normalization purposes was the reaction mixture without fRMsr enzyme. Enzyme activity was calculated using the molar extinction coefficient of NADPH (6220 M1 cm1) and expressed as nmole of oxidized NADPH per min. Km and kcat values were determined by non-linear regression using Prism 5 software (GraphPad). Measurements of the free thiol content of TafRMsr The free Cys content of wild type TafRMsr and mutants were determined using 5,50 -dithiobis(2-nitro)benzoate (DTNB) under denaturing conditions after treatment or no treatment of Met-O, as described elsewhere [17]. Briefly, 10 lM TafRMsr enzyme in a buffer (50 mM sodium phosphate, pH 7.5, and 50 mM NaCl) was treated with or without 20 mM Met-(R,S)-O for 30 min at 37 °C. Sodium dodecyl sulfate (SDS) was added to a final concentration of 10 mM and the enzyme solution was heated for 10 min for 70 °C. DTNB was treated with a final concentration of 300 lM and progress curves of thionitrobenzoate (TNB) production were monitored at 412 nm at 25 °C. The amount of TNB formed was calculated using the extinction coefficient of 13,600 M1 cm1 [17]. Crystallization, data collection, and structure determination

Scheme 1. Catalytic mechanism of type I fRMsr. The catalytic Cys84 attacks the sulfoxide of Met-R-O to form a sulfenic acid intermediate, with concomitant release of Met. The resolving Cys50 then reacts with the Cys84 sulfenic acid to form a disulfide bond, which is reduced by the reductant Trx.

The TafRMsr used for crystallization was concentrated to 10 mg/ml in a buffer (30 mM Tris–HCl, pH 8.0, and 5 mM DTT). The crystals of the native TafRMsr were obtained by the hangingdrop vapor diffusion method by mixing 1 ll protein with 1 ll well solution containing 0.17 M ammonium acetate, 0.085 M sodium

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acetate trihydrate (pH 4.6), 25.5% polyethylene glycol 4000, and 15% glycerol. The mutant C84S TafRMsr was crystallized with a reservoir solution consisting of 60% (v/v) Tacsimate (pH 7.0; Hampton Research). Both native and mutant TafRMsr crystals grew at 20 °C within 2 days. To obtain a substrate-bound form, the crystals of C84S TafRMsr were soaked with the mother liquid containing 10 mM Met-R-O at 22 °C for 1 h. Crystals were mounted and flash-frozen in liquid nitrogen. Xray diffraction data were collected on beamline 5C-SBII at the Pohang Light Source (Pohang, Korea). The data sets were indexed, integrated, and scaled using HKL2000 [18]. The native and substrate-bound TafRMsr crystals diffracted at 2.6 and 2.0 Å, respectively. The detailed data collection statistics are summarized in Table 1. The crystal structure of native TafRMsr was solved by the molecular replacement method with the program MOLREP [19] using the structure of Neisseria meningitidis fRMsr (PDB 3MMH), which shows 36% amino sequence identity with TafRMsr, as a search model [14]. The structure refinement was performed with rigid body refinement using REFMAC, followed by restrained refinement [20]. With iterative cycles of refinement, the structure was manually built using the Coot program [21]. The structure of substrate-bound C84S TafRMsr was determined using the native TafRMsr structure by molecular replacement. The 2Fo-Fc and FoFc residual maps at the active site pocket allowed the construction of Met-R-O binding. Water molecules were added using Phenix [22]. Subsequent refinement was accomplished with REFMAC [20] and Phenix [22], and the refined structures were validated by MOLPROBITY [23]. The final models had an Rfree of 23.9% and an Rfactor of 19.5% at 2.6 Å for the native TafRMsr, and an Rfree of 23.6% and an Rfactor of 19.2% at 2.0 Å for the substrate-bound TafRMsr. The structure refinement data are summarized in Table 1. The atomic coordinates and structure factors have been deposited Table 1 Data collection and refinement statistics.

Data collection Wavelength (Å) Resolution (Å) Space group Unit cell (Å) Observed reflections Unique reflections Redundancy Completeness (%) I/r(I) Rmerge (%)b

Native

Met-R-O complex

0.9788 30–2.6 (2.64–2.6)a H3 a = b = 158.90, c = 194.59 265,551 54,183 4.9 (1.8) 96.2 (73.6) 14.20 (1.4) 8.5 (38.9)

0.9725 30–2.0 (2.03–2.0) P41212 a = b = 120.08, c = 66.91 278,525 33,454 8.3 (5.4) 99.5 (99.4) 40.96 (2.72) 5.9 (44.9)

Refinement Resolution (Å) 28.09–2.6 Rfactor/Rfree (%)c 19.56/23.96 No. of atoms Protein 8912 Water 177 Ligand – R.m.s deviations Bond lengths (Å) 0.009 Bond angles (°) 1.190 2 Mean B values (Å ) Overall 66.16 Protein 66.27 Water 60.31 Ligand – Ramachandran plot (%) Favored regions 97.74 Allowed regions 2.26 Outlier regions 0 a b c

29.83–2.0 19.24/23.64 2311 174 10 0.018 1.859 45.99 45.25 50.51 57.26 98.25 1.75 0

Values in parentheses refer to the highest resolution shell. Rmerge = Rhkl Ri|Ihkli  hIhklii|/Rhkl RihIhklii. Rcryst = Rhkl||Fo|  |Fc||/R|Fo|.

in the Protein Data Bank with the accession codes 4MMN for the native TafRMsr and 4MN7 for the substrate-bound TafRMsr.

Results Biochemical and kinetic properties of TafRMsr TafRMsr, consisting of 141 amino acids, has three Cys residues (Cys15, Cys60, and Cys84). The Cys60 and Cys84 residues are the only two conserved; this enzyme was thus classified as a type II fRMsr (Fig. 1). The purified TafRMsr was in a dimeric form, as judged by size exclusion chromatography. The dimeric property of TafRMsr is consistent with other fRMsrs characterized as dimeric [12,14,15]. The TafRMsr exhibited strict substrate specificity toward free Met-R-O and was unable to reduce dabsylated (peptidyl) Met-R-O, free Met-S-O, or dimethylsulfoxide, like other known fRMsrs [12,14]. To evaluate the role of the three Cys residues in catalysis, each residue was individually mutated to Ser. Catalytic activities and kinetic parameters of the wild type and mutants were then determined using Trx as the reductant. As shown in Table 2, the activity of C84S mutant was not detectable, whereas the other two C15S and C60S mutants exhibited apparent activities. Cys84 residue corresponds to a previously known catalytic residue in type I fRMsrs from S. cerevisiae, N. meningitidis, and S. aureus [14–16]. Together, the data suggested that Cys84 acts as the catalytic residue of TafRMsr. The kcat and Km values of C15S were similar to those of wild type, showing that Cys15 is dispensable for catalysis by TafRMsr. In contrast, the kcat value was dramatically reduced in the C60S mutant (80-fold lower), while the Km value did not significantly change compared to wild type. The data show that Cys60 is critical for the catalytic activity of TafRMsr. The crucial catalytic function of this Cys residue was also reported for the S. cerevisiae fRMsr enzyme [13,16]. Next, the in vivo analysis of activities of wild type TafRMsr and mutants were performed via a growth complementation assay. Yeast expression constructs for the wild type and mutants were generated and separately transformed into a triple msrA/msrB/ fRmsr-deleted S. cerevisiae strain, which is unable to grow in a Met-O medium but can grow in a Met medium. The expression of each construct was confirmed by Western blot analysis (Fig. 2A). As expected, the yeast cells containing an empty vector did not grow in the Met-O medium, but grew in the Met medium (Fig. 2B). Cells expressing wild type TafRMsr grew well in the MetO medium, indicating sufficient reduction of Met-R-O to Met to sustain cell growth. In contrast, cells expressing C84S did not grow in the Met-O medium at all, supporting the hypothesis that Cys84 functions as the catalytic residue in TafRMsr. Cells expressing the C15S mutant showed similar growth to wild type-expressing cells. In cells expressing C60S, growth was not observed during a 3-day incubation but observed at a 7-day incubation. The in vivo fRMsr activities of the wild type and mutant forms correlated well with their in vitro activities. Collectively, the data reveal the roles of the three Cys residues in the catalytic functions of TafRMsr: Cys84 acts as a catalytic residue, Cys60 as a critical residue, and Cys15 as a dispensable residue. The formation of an intramolecular disulfide bond between catalytic and resolving Cys residues is an integral step in the catalytic mechanism of the type I fRMsr enzymes. To investigate whether the resolving Cys is involved in the catalytic functions of the type II TafRMsr enzyme, the free thiol contents of the wild type and different mutants were determined by a reaction with DTNB under denaturing conditions, after treatment or no treatment of Met-O, in the absence of reductant. If there is involvement of a resolving Cys that reacts with the catalytic Cys84 sulfenic acid, two thiol

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Fig. 1. Multiple sequence alignment of type I and II fRMsrs. The catalytic Cys residues in type I and II fRMsr enzymes are highlighted in red and the resolving Cys in type I fRMsr enzymes are highlighted in blue. Another conserved Cys residue is highlighted in green. GenBank accession numbers are as follows: T. acidophilum, 16081903; Dokdonia donghaensis, 86131555; Alkaliphilus metalliredigens, 150389407; Methanothermus fervidus, 312136653; E. coli, 386609218; S. aureus, 421956098; N. meningitidis, 296278577; S. cerevisiae, 549689. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Kinetic parameters of wild-type and mutant forms of TafRMsr. Form

kcat (min1)

Km (mM)

kcat/Km (min1 mM1)

Wild type C15S C60S C84S

16.1 ± 2.3 17.4 ± 1.6 0.2 ± 0.03 ND

0.40 ± 0.17 0.47 ± 0.13 0.33 ± 0.23 –

40.2 37.0 0.6 –

ND, not detectable.

groups in wild type, C15S, or C60S proteins should be lost upon Met-O treatment. However, one thiol group in wild type and C15S and C60S mutants was lost upon Met-O treatment, whereas none was lost for the C84S mutant (Table 3). These data suggest that both Cys15 and Cys60 are not resolving Cys residues; furthermore it suggests no involvement of the resolving Cys in the catalytic mechanism of TafRMsr. Notably, the conserved Cys60 residue also does not function as a resolving Cys in N. meningitidis and S. aureus fRMsrs [14,15]. Antioxidant role of TafRMsr To investigate the protective role of TafRMsr against oxidative stress, yeast cells expressing wild type and mutants, as well as cells containing an empty vector, were tested for sensitivity to H2O2. In the presence of 0.3 mM H2O2, cells expressing the wild type TafRMsr showed increased resistance to peroxide treatment, compared to control cells containing an empty vector (Fig. 3). Cells expressing the C15S mutant also exhibited increased resistance to H2O2-mediated oxidative stress. The resistance was similar to that of the wild type-expressing cells. In contrast, cells expressing both the C84S and C60S mutants showed no resistance to the peroxide treatment. The resistance of the wild type and mutants forms to oxidative stress agreed well with their in vivo and in vitro fRMsr activities. Taken together, the data indicate that TafRMsr can function as an antioxidant to protect cells against oxidative stress. Overall structure of TafRMsr The crystal structure of native TafRMsr was solved at a resolution of 2.6 Å. The TafRMsr crystal belongs to the space group H3

and there are four dimers in an asymmetric unit. The dimer interface region was estimated using the program PISA [24] and its area calculated at 769 Å2 per monomer (10.6% of the total surface area). The dimer interface involves hydrogen bond interactions of residues in helix a1 (Fig. 4A). The carboxylamide group of Gln10 from each subunit interacts with each other, and the side chains of Tyr17 and Lys21 form hydrogen bonds with the backbone of Asp2 and the side chain of Glu5, respectively (Fig. 4A). The structure of a subunit consists of a central b-sheet encompassed by a two-a-helix bundle (a1 and a3) flanking on one side and one small a-helix (a2) on the other side (Fig. 4A). The b-sheet is composed of five antiparallel strands (b2–b1–b5–b4–b3). The strands b2 to b4 are linked by the loop (Lo1, Gly45–Gly58)-a2-b3-loop (Lo2, Tyr73–Thr88) region. Cys60 and Cys84 are present in helix a2 and Lo2, respectively, and a disulfide bond between the two residues was not observed. Cys84 is separated from Cys60 by 5.7 Å. On the other hand, Cys15 is located on the helix a1, which is involved with dimer interaction. Its position is far from Cys60 and Cys84 (12.5 Å and 14.5 Å, respectively), and does not likely interact with these two Cys residues.

Active site of TafRMsr The active site region is organized into a central antiparallel bsheet (b2–b1 and b5–b4), two loops (Lo1 and Lo2), and a short helix (a2) (Fig. 4A). At one side of the active site, three residues (Glu91, Asp107, and Asp109) preceding the b-sheet (b4–b5) constitute a hydrophilic region, whereas the other side forms a hydrophobic region with Trp27 (b1) and Phe85 (Lo2) (Fig. 4B). The main and side chains of residues in the Lo1 are stabilized by hydrogen bond interactions with residues located in two antiparallel bstrands (b1–b2) and the helix a2. The Glu46, His50, and Ile53 on the Lo1 interact with the residues Trp27, Tyr31, and Leu38 preceding the b-strands, respectively, and the two residues, Asp57 and Gly58, also participate in the formation of hydrogen bonds with Ser61 in the helix a2 (Supplementary Fig. S1). These hydrogen bond interactions between the residues in the Lo1 and its surrounding residues involve the stabilization of the loop conformation. On the other hand, the Lo2 containing Cys84, located between the strands b3 and b4, relates to conformational change. The Phe85 displays

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Fig. 2. Growth complementation assay of wild type TafRMsr and its C15S, C60S, and C84S single mutants. Cell growth of triple msrA/msrB/fRmsr-deleted S. cerevisiae cells containing an empty vector (EV) or indicated TafRMsr constructs was analyzed in the Met or Met-O medium via serial dilution (OD600 of 2.5, 0.25, 0.025, and 0.0025). (A) Expression levels of the constructs. Cell lysates were prepared after 3-day culture in the Met medium and subjected to Western blot analysis using anti-His antibodies. (B) Growth complementation assay. Pictures were taken after 3 and 7 days of incubation.

Table 3 Free thiol content in wild type and mutant forms of TafRMsr. Form

Wild type C15S C60S C84S

No. of Cys

3 2 2 2

No. of free thiol measured Without Met-O

With Met-O

Decrease in free thiol

2.5 ± 0.2 1.7 ± 0.2 1.5 ± 0.1 1.7 ± 0.2

1.5 ± 0.2 0.6 ± 0.1 0.6 ± 0.1 1.5 ± 0.1

1.0 ± 0.2 1.1 ± 0.2 0.9 ± 0.1 0.2 ± 0.2

The free thiol content was measured in the denaturing conditions after reaction with or without Met-O in the absence of the reductant.

fewer interactions between the residues of the Lo2 and its surrounding residues, compared with the Lo1. These fewer interactions are related to the flexibility of the Lo2 contributing to conformational changes with substrate interactions and formation of the active site cavity, as explained below.

Structure of TafRMsr in a complex with its substrate

Fig. 3. TafRMsr protects against oxidative stress. The triple msrA/msrB/fRmsrdeleted S. cerevisiae cells containing an empty vector (EV) or indicated TafRMsr constructs (wild type, C15S, C60S, and C84S) were incubated for 2 days in YNBD agar media via serial dilution (OD600 of 2.5, 0.25, and 0.025) with or without 0.3 mM H2O2.

van der Waals interactions with Trp27 in the strand b1 and the rest of the Lo2 shows high flexibility (Supplementary Fig. S1). There are

To obtain the structure of TafRMsr in a complex with its substrate, crystals of the C84S mutant were soaked with free Met-RO and the structure determined at 2.0 Å by molecular replacement analysis using the native TafRMsr structure as a template. The crystal belongs to the space group P41212 with two molecules in a dimeric form in the asymmetric unit. A substrate binds one active site of the dimer (Molecule 2), whereas no substrate binds the other active site of the dimer (Molecule 1) (Supplementary Fig. S2). Instead, several more waters are present in the other active site through hydrogen bond interactions (Fig. 4C). It should be noted that the soaking time of the substrate in crystals might be insufficient for substrate binding to the two active sites.

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Fig. 4. Structure of the TafRMsr. (A) Ribbon representation for the overall structure of the native TafRMsr. Cys15, Cys60, and Cys84 are shown by stick models. The two loops (Lo1, Gly45–Gly58 and Lo2, Tyr73–Thr88) participating in the formation of the active site pocket are in pink and green, respectively. A close-up view represents the dimer interface region. The residues interacting via a hydrogen bond and involved in the dimer formation are shown in stick models. (B) The active site of the native TafRMsr. The residues interacting with several water molecules are depicted by stick models. (C) The active site of the substrate-unbound C84S TafRMsr. The residues interacting with several water molecules are depicted by stick models. (D) A stereo-view of the active site of C84S TafRMsr in complex with Met-R-O. The Met-R-O is depicted as a magenta colored stick model and water molecules as red spheres. The rA-weighted 2Fo-Fc electron density map for the substrate is contoured at 1.0 r in cyan. The interacting residues with Met-R-O are depicted by stick models and the hydrogen bond interactions are represented by black dotted lines. (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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The free Met-R-O is bound in the small active site cavity with solvent accessibility. The substrate is positioned over the central antiparallel b-sheet (b2–b1 and b5–b4) and between the Lo1 and Lo2 (Supplementary Fig. S2). The surrounding residues of the active site participate in substrate binding (Fig. 4D and Supplementary Fig. S3). The oxygens (O1 and O2) of carboxylate of the substrate are hydrogen-bonded to the main peptide chain nitrogens of Leu59 and Cys60, respectively. The NH+3 of the substrate interacts with a water molecule (Wat65), which forms hydrogen bonds with the side chain of Tyr81 and the main chain Leu82 on the Lo2, and also forms a salt bridge with Glu91. Also, another water molecule (Wat58) is hydrogen-bonded to the NH+3 of the substrate and interacts with the sulfoxide oxygen, C84S (Ser84), and Asp109. The sulfoxide moiety of the substrate points toward C84S with a distance of 2.4 Å and the sulfoxide oxygen forms the polar interactions with the side chains of Asp107 and Asp109, and a water molecule (Wat58). The Cb and Cc of the substrate form van der Waals interactions with Cys60. Also, the side chains of Trp27, Tyr31, and Phe85 create a hydrophobic region within the active site, contributing to the stabilization of the e-methyl group of the substrate. Thus, the hydrophilic region of the active site is necessary for substrate specificity, whereas the hydrophobic region plays a significant role in the orientation and binding stabilization of the substrate. Conformational change in the active site pocket of TafRMsr The structure of substrate-bound C84S TafRMsr was compared with those of the native and substrate-unbound C84S forms to describe conformational changes of the structure for substrate recognition (Fig. 5A). As mentioned, a disulfide bond between Cys84 and Cys60 is not observed in the native TafRMsr, indicating a reduced form. There appears to be little shift of these two Cys residues between the reduced native TafRMsr and the substrateunbound and substrate-bound forms of C84S (Fig. 5A). Several other water molecules, instead of the substrate, are present in the active site pockets of the native TafRMsr and substrateunbound C84S forms. These water molecules interact with Cys60, Glu91, Asp107, and Asp109 residues (Fig. 4B and C). Interestingly, the Lo1 and Lo2 regions (containing four residues Asn54, Asp57, Tyr81, and Leu82) are different between the native TafRMsr and the substrate-unbound C84S form. Notably, these loops play an important role in the formation of the active site pocket, as discussed below. Thus, the structure of the substrate-unbound C84S TafRMsr can be regarded as an intermediate active site structure immediately preceding substrate binding into the active site. Structural superimposition of the three TafRMsr forms indicates significant differences in the Lo1 and Lo2 regions (Fig. 5A). Movements of these regions cause the shifts of particular residues critical to the formation of an ‘open’ active site pocket from a ‘closed’ form. For substrate binding in the active site, Asp57 in the Lo1, and Tyr81 and Leu82 in the Lo2, are the most displaced residues and shift in opposite directions by 6.6, 7.1, and 7.2 Å, respectively. Also, there is a shift of 3.3 Å in the side chain of Asn54 located in the Lo1. The conformational change of the TafRMsr active site pocket occurs by the shift of these four residues involved in the formation of the active site pocket. Interestingly, the native reduced TafRMsr reveals a ‘closed’ active site pocket, with Asn54 and Leu82 residing at a distance of 7.0 Å, facing each other in the direction of the active site cavity (Fig. 5B). Furthermore, the substrate-unbound C84S TafRMsr forms an ‘open’ active site cavity by the shift of the two Asn54 and Leu82 residues in the opposite direction of the active cavity with a distance of 16.6 Å (Fig. 5C). In addition to the shift of the Asn54 and Leu82, the shift of Asp57 to the outside of the active site pocket opens the active site. However, after substrate binding, the active site pocket becomes ‘narrow’ because the Asp57 residue is re-shifted forward into the active site pocket (Fig. 5D). The Tyr81

residue also moves to the inside of the active site pocket and stabilizes substrate binding by interacting with the substrate nitrogen via a water molecule (Wat65) (Fig. 5D).

Discussion The present study biochemically and structurally characterized a type II fRMsr from T. acidophilum. The TafRMsr possesses three Cys residues (Cys15, Cys60, and Cys84), of which Cys60 and Cys84 are conserved. The in vitro and in vivo activity assays indicate that Cys84 is the catalytic residue of TafRMsr. The C84S mutant showed no detectable activity in the enzyme assay, whereas the C15S and C60S mutants had apparent activities. Moreover, yeast cells expressing the C84S mutant did not grow in Met-O medium. The catalytic function of Cys84 was also supported by our structural analysis. Based on the biochemical and structural analyses, a catalytic mechanism can be proposed for the type II TafRMsr, in which no intramolecular disulfide bond formation is involved (Scheme 2). The crystal structures of TafRMsr in this study also illuminate how the substrate accesses and binds to the active pocket through the interactions of active site residues and conformational changes. Interactions between the substrate and residues within the active site are major factors for the orientation and stabilization of the substrate. The oxygen of the sulfoxide is positioned in a hydrophilic region. It has direct interactions with the residues Asp107 and Asp109, as well as with Wat58 (Fig. 4D and Supplementary Fig. S3). These acidic residues are significant residues for the binding and stabilization of the sulfoxide oxygen in the active site. These interactions also help orientate Cys84 for its attack on the sulfoxide moiety of the substrate. The interactions of the b- and c-methylene groups of the substrate with Cys60 and Ile53 (via van der Waals interactions) and the location of the e-methyl group of the substrate in the hydrophobic region (consisting of Trp27, Tyr31, and Phe85 residues) play major roles for conformational stabilization, similar to type I N. meningitidis fRMsr [14]. Finally, the carboxylate group of the substrate is stabilized by interactions with the main chains of Leu59 and Cys60, which form an oxyanion hole. The orientation of the substrate in the active site is controversial in type I fRMsr enzymes [14,15]. The substrate orientation in TafRMsr is similar to that in N. meningitidis fRMsr [14]. Collectively, the structural analysis not only identifies Cys84 as the catalytic residue but also how the TafRMsr specifically recognizes the R isomer by showing the specific orientation of the substrate that cannot be reversed in the active site. Among the three Cys residues of TafRMsr, the Cys15 residue is positioned on the helix a1 far away from the active site, whereas the two conserved Cys60 and Cys84 at the helix a2 and Lo2, respectively, are located within the small active site pocket. However, a disulfide bond between Cys84 and Cys60 is not formed in the crystal structure of the native TafRMsr. Four crystal structures of type I fRMsr enzymes from E. coli, S. cerevisiae, N. meningitidis, and S. aureus have been solved to date [12,14,15,25], with or without substrate. Notably, an oxidized form with a disulfide bond between the catalytic and resolving Cys has been obtained from all four type I fRMsr structures. These data suggest that in type I fRMsr enzymes the formation of a disulfide bond between catalytic and resolving Cys is relevant during protein purification and crystallization. To obtain the structure of TafRMsr, which includes the formation of the disulfide bond between Cys84 and Cys60, protein purification and crystallizations were additionally performed in the absence of DTT. However, disulfide bond formation between these two Cys residues was not observed in the crystals. The TafRMsr structure shows that Cys60 is involved in the stabilization of the band c-methylene and carboxylate groups of the substrate. Although the Cys60 residue is crucial for the catalytic activity of

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Fig. 5. Structural comparison and conformational changes of native TafRMsr and substrate-unbound and substrate-bound C84S TafRMsr. (A) The structural superimposition of the native TafRMsr (cyan) and substrate-unbound (yellow) and substrate-bound (blue) C84S TafRMsr. Each structure is represented in ribbon models and the significantly different regions shown in the active sites are illustrated close-up in a black outlined square. The shifted residues are depicted by stick models and their movement directions are shown by arrows. (B–D) Molecular surface models in the same view and colors as in (A). (B) The native TafRMsr. The active site pocket is closed by the residues Asn54, Asp57, and Leu82. (C) The substrate-unbound C84S TafRMsr. Its active site pocket is opened by the shift of Asn54, Asp57, and Leu82. This structure may indicate an intermediate state immediately preceding recognition of the substrate. (D) The substrate-bound C84S TafRMsr. The Asp57 is re-shifted and Tyr81 moves to the inside of the active site pocket and interacts with Wat65 to stabilize substrate binding. The active site pocket becomes narrow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

TafRMsr, our structural analysis suggests that this residue does not have the resolving function. This conclusion is consistent with the free thiol content data, showing that only one thiol group for the wild type TafRMsr and C15S mutant was lost upon Met-O treatment. Conformational changes in the active site cavity are required for substrate recognition. In the absence of free Met-R-O, the active site pocket is closed through Asn54 and Leu82 facing each other in a forward direction within the active site cavity (Fig. 5B). However, in the presence of the substrate, with the movement of the Lo1 and Lo2, these Leu82 and Asn54 residues, as well as Asp57, are shifted in the opposite direction in the active site cavity; consequently the active site cavity is open for substrate access (Fig. 5C). After substrate binding, the active site pocket becomes narrow by the reshifting of Asp57 forward into the active site cavity (Fig. 5D). In addition, the shifting of the side chain of Tyr81 toward the substrate nitrogen helps the stability of substrate binding. Tyr81 is a conserved residue in type II fRMsrs, which is replaced with His in type I enzymes (Fig. 1). This Tyr residue is thus considered as an important residue which contributes to the formation of the narrowed active site pocket and stabilization of the substrate binding. The narrowed active site pocket perhaps prevents the accessibility of another substrate in the active sites, before the release of the product Met. These conformational changes differ from those of type I S. aureus fRMsr [15], which is initially ‘open’ in the reduced form and ‘closed’ in the substrate-bound form.

The structural differences and similarities between the two types of fRMsr enzymes were identified. The overall structure of the type II TafRMsr is similar to that of type I N. meningitidis and S. aureus fRMsr enzymes (with an r.m.s.d of 0.75 and 0.97 Å, measured with 82 and 98 Ca atoms, respectively), except that the type I fRMsr enzymes have an additional helix at the N-terminal region compared to the type II TafRMsr. However, the dimerizations of the type II TafRMsr versus type I fRMsr enzymes are quite different. Residues on the opposite directional helix a1 in each subunit of type II TafRMsr are involved in dimerization by hydrogen bond interactions (Fig. 4A), whereas the type I S. aureus fRMsr forms a dimer interface via interactions among residues on the second helix and a loop between b2 and b3 [15]. The catalytic Cys84 residue of TafRMsr is situated at position similar to the catalytic Cys of type I fRMsr enzymes (Cys109 for N. meningitidis fRMsr and Cys102 for S. aureus fRMsr) and the Cys60 residue is located at the same position as another conserved Cys of type I fRMsr enzymes (Cys85 for N. meningitidis fRMsr and Cys78 for S. aureus fRMsr) (Supplementary Fig. S4). Interestingly, His50 is positioned at the site of the resolving Cys residue of type I fRMsr enzymes (Cys75 for N. meningitidis fRMsr and Cys68 for S. aureus fRMsr). The residue His50 is located at a distance of 4.0 Å from Cys84. Notably, the His50 is a conserved residue in type II fRMsr enzymes that replaces the resolving Cys found in type I fRMsr enzymes (Fig. 1). Wat58 interacts with the sulfoxide oxygen and NH+3 of the substrate, the main chain of C84S, and the side

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Scheme 2. Proposed catalytic mechanism of TafRMsr. Asp109 protonates and acts as a proton transfer from the catalytic Cys84 thiol to the sulfoxide (step 1). Asp107 may act as an alternative proton transfer to the sulfoxide. Asp109, Wat58, and Asp107 participate in stabilizing the polarized sulfoxide oxygen. Wat58 also interacts with Asp109. The nucleophilic attack of the catalytic Cys84 thiolate to the sulfur of the sulfoxide moiety occurs and the sulfurane transition state is formed (step 2). The rearrangement of the transition state gives rise to the formation of the sulfenic acid on Cys84, with release of the product Met (step 3). The Cys84 sulfenic acid is directly reduced by the reductant Trx (step 4).

chain of Asp109. It resembles a water molecule (Wat99) in type I N. meningitidis fRMsr, which interacts with the sulfoxide oxygen and participates in stabilization of the sulfurane transition state [14]. Two negatively charged invariant residues Asp107 and Asp109 in TafRMsr show the hydrogen-bond interactions with the oxygen atom of the sulfoxide (2.6 and 2.7 Å, respectively). The two Asp107 and Asp109 residues are located 5.7 and 5.5 Å from C84S and Cys84, respectively. Considering the distances of Asp107 and Asp109 from the sulfoxide oxygen and the catalytic Cys84, either of the two Aps residues may act as a proton transfer from the catalytic Cys to the oxygen of the sulfoxide. Asp109 is suggested to be the proton transfer in type I N. meningitidis fRMsr [14]. Based on the structural and biochemical characterization, a model for the catalytic mechanism of the type II TafRMsr is suggested, in which intramolecular disulfide bond formation is not involved (Scheme 2). The polarized sulfoxide moiety is stabilized by the interactions with Asp109, Asp107, and Wat58. These interactions lead to the nucleophilic attack of the catalytic Cys84 to the sulfur of free Met-R-O. A sulfurane intermediate/transition state is formed and its rearrangement gives rise to the formation of a sulfenic acid intermediate. The Cys84 sulfenic acid can be directly reduced by the reductant Trx. The significant difference in the catalytic mechanism of type II TafRMsr, compared to that of type I fRMsr enzymes, is the absence of an intramolecular disulfide bond involving a resolving Cys. This non-resolving Cys involvement mechanism has also been suggested for some MsrBs, including mammalian MsrB2 and MsrB3, which can be directly reduced by Trx [26,27]. Acknowledgments We thank the staff of beamline 5C-SBII at the Pohang Light Source (Korea) for technical support, the staff at the Korea Basic

Science Institute (KBSI, Daejeon, Korea) for the use of a mosquito crystallization robot, and the members at beamline BL-17A of the Photon Factory for their assistance with data collection (2012G189; Tsukuba, Japan). This work was supported by grants from the National Research Foundation of Korea (2011-0028166 and 2013R1A1A2008404). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2014.07.009. References [1] H. Weissbach, F. Etienne, T. Hoshi, S.H. Heinemann, W.T. Lowther, B. Matthews, G. St John, C. Nathan, N. Brot, Arch. Biochem. Biophys. 397 (2002) 172–178. [2] M. Kantorow, J.R. Hawse, T.L. Cowell, S. Benhamed, G.O. Pizarro, V.N. Reddy, J.F. Hejtmancik, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9654–9659. [3] J. Moskovitz, E. Flescher, B.S. Berlett, J. Azare, J.M. Poston, E.R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 14071–14075. [4] F. Cabreiro, C.R. Picot, M. Perichon, J. Castel, B. Friguet, I. Petropoulos, J. Biol. Chem. 283 (2008) 16673–16681. [5] G.H. Kwak, J.R. Kim, H.Y. Kim, BMB Rep. 42 (2009) 113–118. [6] H.Y. Kim, Antioxid. Redox Signal. 19 (2013) 958–969. [7] J. Moskovitz, Biochim. Biophys. Acta 1703 (2005) 213–219. [8] H.Y. Kim, V.N. Gladyshev, Biochem. J. 407 (2007) 321–329. [9] V.S. Sharov, D.A. Ferrington, T.C. Squier, C. Schoneich, FEBS Lett. 455 (1999) 247–250. [10] G.H. Kwak, K.Y. Hwang, H.Y. Kim, Arch. Biochem. Biophys. 527 (2012) 1–5. [11] R. Grimaud, B. Ezraty, J.K. Mitchell, D. Lafitte, C. Briand, P.J. Derrick, F. Barras, J. Biol. Chem. 276 (2001) 48915–48920. [12] Z. Lin, L.C. Johnson, H. Weissbach, N. Brot, M.O. Lively, W.T. Lowther, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 9597–9602. [13] D.T. Le, B.C. Lee, S.M. Marino, Y. Zhang, D.E. Fomenko, A. Kaya, E. Hacioglu, G.H. Kwak, A. Koc, H.Y. Kim, V.N. Gladyshev, J. Biol. Chem. 284 (2009) 4354–4364. [14] A. Gruez, M. Libiad, S. Boschi-Muller, G. Branlant, J. Biol. Chem. 285 (2010) 25033–25043.

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