Archives of Biochemistry and Biophysics 545 (2014) 1–8
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Structural analysis of 1-Cys type selenoprotein methionine sulfoxide reductase A Eun Hye Lee a, Geun-Hee Kwak b, Moon-Jung Kim b, Hwa-Young Kim b,⇑, Kwang Yeon Hwang a,⇑ a b
Division of 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 22 October 2013 and in revised form 23 December 2013 Available online 8 January 2014 Keywords: Methionine sulfoxide reductase MsrA Selenoprotein Selenocysteine Catalysis Clostridium
a b s t r a c t Methionine sulfoxide reductase A (MsrA) reduces free and protein-based methionine-S-sulfoxide to methionine. Structures of 1-Cys MsrAs lacking a resolving Cys, which interacts with catalytic Cys, are unknown. In addition, no structural information on selenocysteine (Sec)-containing MsrA enzymes has been reported. In this work, we determined the crystal structures of 1-Cys type selenoprotein MsrA from Clostridium oremlandii at 1.6–1.8 Å, including the reduced, oxidized (sulfenic acid), and substrate-bound forms. The overall structure of Clostridium MsrA, consisting of ten a-helices and six b-strands, folds into a catalytic domain and a novel helical domain absent from other known MsrA structures. The helical domain, containing five helices, tightly interacts with the catalytic domain, and is likely critical for catalytic activity due to its association with organizing the active site. This helical domain is also conserved in several selenoprotein MsrAs. Our structural analysis reveals that the side chain length of Glu55 is critical for the proton donor function of this residue. Our structures also provide insights into the architecture of the 1-Cys MsrA active site and the roles of active site residues in substrate recognition and catalysis. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The oxidation of methionine to methionine sulfoxide readily occurs due to cellular reactive oxygen species and may induce significant alterations in protein structure and function [1,2]. However, this modification can be reversed by enzymatic reactions [3]. Methionine sulfoxide reductases (Msrs) are the enzymes that catalyze the reduction of methionine sulfoxide in a stereospecific manner [4,5]. MsrA is specific for the S-form of methionine sulfoxide, whereas MsrB only acts on the R-form. Selenoproteins, selenocysteine (Sec)-containing proteins, are found in all three kingdoms of life [6–8]. Selenoprotein forms of Msrs have been identified in many organisms, from bacteria to humans [9]. The Sec residue located at the catalytic sites of selenoprotein Msrs provides higher catalytic activity compared to Sec-to-Cys mutants or Cys-containing homologs [10–13]. Clostridium oremlandii (strain OhILAs), particularly rich in selenoproteins, is a strictly
⇑ Corresponding authors. Address: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchungno, Namgu, Daegu 705-717, Republic of Korea. Fax: +82 53 629 7093 (H.-Y. Kim). Address: Division of 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). E-mail addresses: [email protected] (H.-Y. Kim), [email protected] (K.Y. Hwang). 0003-9861/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.12.024
anaerobic, Gram-positive bacterium that contains the selenoprotein MsrA [13]. This selenoprotein MsrA consists of 209 residues and has a Sec residue within its active site, but contains no Cys residues. This selenoenzyme appears to be among the most efficient MsrA catalysts known [12,13]. The enzymatic activity of the selenoprotein MsrA is 20-fold higher than its Sec-to-Cys form; thus Sec is critical in the catalysis of this selenoenzyme [13]. However, the Sec-to-Cys form also has an MsrA activity comparable to mammalian Cys-containing MsrA [13]. The conserved active site sequence of MsrA is GCFW(G/H) and includes the catalytic Cys [14]. The catalysis of MsrA is based on common sulfenic acid chemistry [15,16]. MsrAs can be divided into three groups based on the resolving Cys, which interacts with catalytic Cys to form an intramolecular disulfide bond: 3-Cys MsrAs, such as the enzymes from Escherichia coli and Bos taurus, have two resolving Cys [15,17,18]; 2-Cys MsrAs, such as the enzymes from Mycobacterium tuberculosis and Streptococcus pneumoniae [19,20], include a single resolving Cys; and 1-Cys MsrAs, such as the MsrA from Synechocystis sp., contain no resolving Cys [21]. The structures of 3-Cys or 2-Cys MsrAs have been well described [17,19,20,22–25], but no structural information on 1-Cys MsrA has been reported yet. Furthermore, the structure of Sec-containing MsrA is unknown. In this work, the crystal structures of 1-Cys type selenoprotein MsrA from C. oremlandii, in which Sec was replaced with Cys, are
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described for the first time. The structures of reduced, oxidized (sulfenic acid), and substrate-bound forms (all representing structural redox states of the enzyme) have been resolved. The overall structure contains a novel helical domain with five helices. This helical domain interacts with the catalytic domain and is essential for catalytic activity. Also, our data provide structural insights into the roles of active site residues in substrate recognition and catalysis, and conformational changes in the active site pocket based on the redox state of the enzyme. Materials and methods
length of 1.0000 Å, and processed using the HKL2000 package [26]. The crystals belonged to the trigonal space group P3221 in a hexagonal setting with unit cell dimensions of a = b = 80.3 Å, c = 66.9 Å, and contained one molecule in an asymmetric unit. The variants C16S, E55A, and E55D were all treated with 10 mM free methionine-(R,S)-sulfoxide at 25 °C for 30 min prior to crystallization. The crystallization conditions for the variants were similar to that for the Sec-to-Cys MsrA. The data were collected on beamline BL1A at the Photon Factory (Tsukuba, Japan) at a wavelength of 1.0000 Å, and processed using the HKL2000 package. A summary of data collection statistics is provided in Table 1.
Cloning and protein purification
Crystal structure determination and refinement
The sequence of the pET-based C. oremlandii MsrA (CoMsrA) was previously described [13]. Sequences encoding the C16S, E55A, and E55D forms were generated by site-directed mutagenesis using pET-CLOS-MsrA/U16C (Sec-to-Cys form) as a template. The sequence lacking the helical domain (residues 1–144) was PCR-amplified and inserted into the NdeI/XhoI sites of pET21b (Novagen). All constructs included a C-terminal His-tag. Plasmids were introduced into E. coli BL21(DE3) star cells, and recombinant cells were grown in LB medium containing 50 lg/ml ampicillin at 37 °C until the OD600 reached 0.6. The heterologous expression of the recombinant protein was induced by the addition of 0.3 mM isopropyl thiogalactoside (IPTG), after which the cultures were incubated at 18 °C for 16 h. Cells were harvested and the cell pellet lysed by sonication in ice-cold 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 4 mM MgCl2, and 5 mM imidazole. After centrifugation at 17,940g for 50 min, the supernatant was loaded onto a HisTrap column equilibrated in the same lysis buffer. An imidazole gradient (10–500 mM) was applied to the protein-bound HisTrap column to elute the protein. The eluate was concentrated to 2 ml by an Amicon (Millipore; Ultracel regenerated cellulose membrane, MWCO 10 K) and then separated by gel filtration chromatography through a Superdex 200 column in 10 mM Tris–HCl (pH 8.0) and 100 mM NaCl. The >98% purity of CoMsrAs was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) analysis, and the protein solution was concentrated to approximately 10 mg/ml for crystallization.
The crystal structure of CoMsrA was determined by molecular replacement using the structure of E. coli MsrA (EcMsrA, PDB ID: 1ff3) as a search model [23] with the software package Molrep implemented in the CCP4i suite [27]. Further model building was performed using the Coot program [28] and refinement was performed with CCP4 refmac5 and PHENIX [29]. The final model was validated using PROCHECK [30] and had an Rcryst of 17.9% and an Rfree of 21.0%. The structures of the variants were determined using the refined CoMsrA structure by molecular replacement and refinements were similarly performed. A summary of statistics related to structure refinement is provided in Table 1.
MsrA assay and kinetic analysis The MsrA reaction mixture (100 ll) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 20 mM dithiothreitol (DTT), 0.05–0.8 mM dabsyl-methionine-S-sulfoxide, and 1–30 lg purified MsrAs. The reaction was carried out at 37 °C for 30 min, and its product (dabsyl-Met) was detected by high-performance liquid chromatography. Km and kcat values were determined by non-linear regression using Prism 5 software (GraphPad). Crystallization and data collection Due to the low expression level of selenoprotein, the Sec-to-Cys form was used for crystallization. The initial screening of CoMsrA was achieved using the sitting-drop vapor-diffusion method with crystallization conditions (Hampton Research). Crystals suitable for analysis were obtained by the hanging-drop vapor-diffusion method at 20 °C by combining 1.5 ll protein with 1.5 ll precipitation solution. The best crystals were obtained from a reservoir solution consisting of 0.1 M MES (pH 6.5), 30% (w/v) polyethylene glycol monomethyl ether 5000, and 0.2 M ammonium sulfate. Crystals were snap-frozen in liquid nitrogen and subjected to Xray diffraction. A 1.8 Å resolution native data set was collected on beamline 4A at Pohang Light Source (Pohang, Korea) at a wave-
Results and discussion Overall structure of CoMsrA The native CoMsrA carries a Sec residue within its active site. For structure determination, the Sec-to-Cys form of the enzyme was used due to the low expression of the selenoprotein form. The CoMsrA crystal structure was determined at 1.8 Å resolution. One molecule was included in each asymmetric unit, which comprised residues 6–209 and included one additional Leu residue from the His tag (LEHHHHHH) of the recombinant protein. The five N-terminal residues were disordered. The oligomeric state of CoMsrA in solution was also investigated by analytical ultracentrifugation (Supplementary Fig. S1). The result indicated that CoMsrA is a monomeric protein. The monomeric state of CoMsrA is consistent with the fact that other MsrAs characterized so far are monomeric. The overall structure of CoMsrA, comprised of ten a-helices and six b-strands, folded into a catalytic domain (residues 6–144) and an additional helical domain (residues 145–209) (Fig. 1). The catalytic domain consists of a rolled mixed b-sheet, with the exterior side surrounded by five a-helices (a1–a5). The b-sheet is formed by four anti-parallel (b1–b3–b2–b6) and two short parallel (b4– b5) b-strands. The N- and C-terminal sides of the b-sheet are composed of loop regions. The connecting loops (L1 and L2) on the Cterminal side of the b-sheet are slightly longer than those on the opposite side and participate in the formation of the active site. The catalytic Cys16 residue is located on the loop between the b1 strand and the a1 helix. The helical domain consists of five ahelices (a6–a10) that form a four-helix bundle with a short a-helix insertion between the a6 and the a8 helix. Structural comparison of CoMsrA with EcMsrA The EcMsrA is a 3-Cys MsrA that contains two resolving Cys residues (Cys198 and Cys206) in a C-terminal extension. The structure of EcMsrA features extended N-terminal and C-terminal regions surrounding the core domain [23]. The N-terminal region is not essential for catalytic activity, but the C-terminal region provides
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E.H. Lee et al. / Archives of Biochemistry and Biophysics 545 (2014) 1–8 Table 1 Statistics on crystallographic analyses.
a
Sec-to-Cys
C16S
E55A
E55D
PDB ID code
4LWJ
4LWK
4LWL
4LWM
Data collection Space group Asymmetric unit
P3221 Monomer
P32 Dimer
P3221 Monomer
P3221 Monomer
Cell parameters a, b, c (Å) Resolution (Å) Rsym I/rI Completeness (%) Redundancy
80.3, 80.3, 66.9 1.80 (1.83–1.80)a 0.048 (0.221) 32.8 (3.7) 98.0 (97.9) 2.9 (2.3)
81.0, 81.0, 66.7 1.80 (1.83–1.80) 0.060 (0.207) 59.3 (8.2) 99.4 (97.4) 3.3 (2.5)
81.2, 81.2, 66.9 1.60 (1.63–1.60) 0.066 (0.433) 38.3 (2.3) 98.4 (95.2) 3.5 (2.2)
80.8, 80.8, 66.9 1.80 (1.83–1.80) 0.080(0.333) 19.8 (2.2) 97.1 (91.5) 3.0 (1.7)
Refinement No. of reflections Rwork/Rfree (%)
27,101 17.9/21.0
45,156 17.1/20.6
33,211 19.7/23.4
22,944 17.9/22.3
No. of atoms Protein Ligand/ion Water
1641 9 215
3298 18 414
1637 4 155
1639 14 157
B-factors (Å2) Protein Ligand/ion Water
20.569 18.128 25.723
24.616 31.872 34.483
28.708 27.532 37.617
16.381 39.614 24.340
R.m.s deviations Bond lengths (Å) Bond angle (°)
0.007 1.016
0.007 1.003
0.007 1.019
0.015 1.487
Ramachandran plot (%) Most favored Allowed Disallowed
98.5 1.5 0
98.0 2.0 0
98.5 1.5 0
98.0 2.0 0
Values for the highest resolution shell are given in parentheses.
The overall structure of the CoMsrA catalytic domain is similar to EcMsrA, and the catalytic Cys residues, Cys16 in CoMsrA and Cys51 in EcMsrA, coincide (Supplementary Fig. S2). The CoMsrA shares the conserved active site residues with EcMsrA but it shows structural differences near the active site. There are two long loops, L1 (residues 39–53) and L2 (residues 79–92), contributing to formation of the active site. In contrast, EcMsrA contains a helix in the corresponding L1 region and a short b-hairpin in the corresponding L2 region. The short b-hairpin contains an Asp129 residue that contributes to substrate binding by interacting with the backbone nitrogen of the substrate [22]. This residue is conserved in 3-Cys or 2-Cys MsrAs, and the substitution of the Asp129 to Ala leads to a 16-fold increase in Km value [32]. However, the highly conserved Asp residue is absent in CoMsrA. The absence of the conserved Asp in CoMsrA may cause a relatively higher Km value for this enzyme, as characterized previously [13] and in this study (Table 2). Fig. 1. The overall structure of CoMsrA. The structure comprises a 144-residue catalytic domain (pale cyan) and a 65-residue helical domain (red) arranged into 5 helices. The former includes all the catalytic residues. The catalytic C16 residue is represented by a stick model. The specific loop regions L1 and L2 are colored yellow and orange, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the two resolving Cys residues required for thioredoxin-dependent regeneration [18,31]. This flexible C-terminal region, including the resolving Cys residues, is absent from 1-Cys CoMsrA; instead, its catalytic domain is followed by the distinct helical domain (as mentioned above). Other MsrA homologs similar to EcMsrA in structure (e.g., from B. taurus, M. tuberculosis, and Populus trichocarpa) do not include a homolog of the CoMsrA helical domain [17,19,24]. The superposition of the CoMsrA structure on EcMsrA produced a root mean square deviation (r.m.s.d.) of 1.1 Å over 136 Ca atoms.
Interactions between the helical domain and catalytic domain The helical domain has tight interactions with the catalytic domain using the a8 helix located between the catalytic domain a1 and a5 helices (Fig. 2). The hydrophobic stack of Trp18 and Table 2 Kinetic parameters of Sec-to-Cys MsrA and its mutants.
a
1
Form
kcat (min
)
Sec-to-Cys E55A E55D DH-domaina
160 ± 30 0.07 ± 0.015 0.19 ± 0.06 0.26 ± 0.09
Km (mM)
kcat/Km (min
1.92 ± 0.48 0.75 ± 0.28 5.29 ± 1.85 2.54 ± 1.12
83.3 0.09 0.03 0.10
1
DH-domain is a truncated form of MsrA lacking the helical domain.
mM
1
)
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Tyr140 on a1 and a5 helices, respectively, closely holds these helices and contributes to active site formation. Tyr141 on the a5 helix is vertically stacked on these three helices at their converging point. The residues Thr166, Arg170, and Asn172 on the a8 helix contribute to interactions with the a1 and a5 helices. The Od atom of Asn172 forms hydrogen bonds with the amide nitrogen of Tyr141 and Leu142 on the a5 helix. The NH1 and NH2 atoms of Arg170 form hydrogen bonds with the backbone oxygens of Gly25 and Ile27 on the a1 helix and Val30 in the following loop. The Oc atom of Thr166 forms a hydrogen bond with the backbone oxygen of Val31 in the following loop.
Essential role of the helical domain for activity The role of the helical domain was explored by measuring the catalytic activity of a helical domain-truncated form comprising only residues 1–144. In a previous study, an EcMsrA truncated form (containing residues 42–194), which corresponds well to the structure of the helical domain-truncated CoMsrA, showed similar activity to the wild-type in the presence of DTT [18], indicating that the EcMsrA C-terminal region is only required for thioredoxin-dependent regeneration. Due to the high structural similarity of the CoMsrA catalytic domain to the truncated EcMsrA (r.m.s.d. 1.1 Å), the form of CoMsrA deleted in its helical domain was expected to have significant activity in the DTT-dependent condition. However, its catalytic activity was almost completely abolished in the presence of DTT, showing 800-fold lower catalytic efficiency (kcat/Km) compared to the full-length form (Table 2). These data indicate the requirement of the helical domain for catalysis. The helical domain is closely associated with the catalytic domain, interacting with the latter’s a1 and a5 helices via the a8 helix (Fig. 2). Notably, the hydrophobic stack of Trp18 and Tyr140 on the a1 and a5 helices, respectively, contributes to active site formation (Fig. 3A). Thus, the interaction of the helical domain with the catalytic domain may be critical for maintaining active
site organization for catalysis. This interaction might also affect overall folding of the catalytic domain. Further studies will be needed to understand the functional implications of the domain interactions. Conservation of the helical domain in selenoprotein MsrAs To examine the conservation of the helical domain in selenoprotein MsrAs, a multiple sequence alignment was performed (Supplementary Fig. S3). To date, eight selenoprotein MsrA sequences have been reported at UniProt database and may be grouped into two subclasses with respect to the length of the Cterminal region. Two have a short C-terminal tail (10 residues) and are grouped into ‘subclass I’. The other six, including CoMsrA, contain a C-terminal extension (65 residues) and are grouped into ‘subclass II’. The subclass I proteins contain a conserved GUFWH motif and two Cys residues at positions 51 and 88 (numbering based on CoMsrA sequence) that do not serve as a resolving Cys [12]. All subclass II proteins contain the conserved GUFWG motif with no Cys residues in the entire sequence, but one MsrA with two Cys in its C-terminal extension region. These C-terminal extension domains have been predicted to form three to five a-helices by secondary structure prediction using Xtalpred [33]. Overall, the data suggest that the helical domain is conserved in subclass II selenoprotein MsrAs. CoMsrA active site and substrate binding Despite some structural differences around the active site, the architecture of the CoMsrA active site is similar to other MsrA homologs [17,19,23]. A hydrophobic pocket formed by Phe17 and Trp18 in the conserved GCFWG motif occupies one side of the site, while the Tyr47, Glu55, and Tyr90 side chains create a hydrophilic region on the other side (Fig. 3A). Interestingly, the bulge in electron density corresponding to the Cys16 side chain is recognizable
Fig. 2. Interaction of the helical domain with the catalytic domain. Interactions between the helical (red) and catalytic domain (pale cyan) are represented. The helical domain a8 closely interacts with the catalytic domain a1 and a5 through hydrogen bonds. The interacting residues are displayed in a stick model, and hydrogen bonds between the residues are indicated as dashed lines. The two interacting regions are magnified in right squares a and b. (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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Fig. 3. The active site of CoMsrA and its substrate recognition. The active site residues are represented by a stick model, and an electron density map relevant for binding is shown. (A) Active site of the Sec-to-Cys CoMsrA. A water molecule (Wat1, red sphere) is bound at the active site, where it interacts with four residues, including the C16 sulfenic acid form. The bulge of electron density is equivalent to the C16 Od sulfenic oxygen. (B) Active site of the C16S variant. Three water molecules (Wat1, Wat2, and Wat3) are bound at the active site of the C16S variant. (C) Active site of the E55A variant. The Q89 side chain is twisted away from the active site due to loss of the interaction with the E55 side chain. The C16 side chain is present in a sulfenic acid form, similar to Sec-to-Cys. (D) Active site of the substrate-bound E55D variant. The substrate methionine-S-sulfoxide (Met-S-O, gray stick model) is observed at the active site. The electron density associated with the methionine sulfoxide terminal side chain is strong, but the backbone electron density is weak. The electron density for the aromatic ring of Y47 residue is weak. The sulfoxide oxygen atom is stabilized via Q89 and Y90 and the sulfur atom interacts with the C16 sulfur atom. The 2Fo-Fc map contoured at 1.5r shows the electron density for bound molecules at the active site. (E) A stereoview of the active site of substrate-bound E55D. The active site residues (magenta) and substrate (gray) are displayed in the stick model. Met-S-O, methionine-S-sulfoxide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
as the sulfenic acid hydroxyl group. Thus, this structure represents an sulfenic acid form that has also been reported in Neisseria meningitidis MsrA (NmMsrA, PDB ID: 3bqg) [22]. The Od sulfenic oxygen atom of Cys16 lies 3.5 Å from a water molecule (Wat1). Wat1 is stabilized in the active site by its interaction with the Tyr47 (2.7 Å), Glu55 (2.6 Å), and Tyr90 (2.7 Å) side chains. Within the active site, the Gln89 Ne2 atom interacts with the Glu55 Oe2 atom (2.7 Å in distance), while its side chain extends outside the active site in the E. coli and bovine homologs [17,23]. On the opposite side, a highly conserved hydrophobic pocket is formed through a stable p–p interaction between the Trp18 indole ring and the Tyr140 aromatic side chain and by the interaction of the Trp18 indole ring with the His137 side chain. The nature of the CoMsrA active site was further investigated through a comparison of the structures of the three single residue variants (C16S, E55A, and E55D) in the active site (Fig. 3B–E). The C16S variant was catalytically inactive, while the E55A and E55D mutants were almost inactive (Table 2). The kcat values of E55A and E55D mutants were 2300- and 840-fold lower, respectively, than that of Sec-to-Cys MsrA. To obtain a substrate-bound form structure, the variant CoMsrAs were each co-crystallized with the
substrate methionine sulfoxide. However, an electron density map consistent with the substrate was only produced by the E55D variant (Fig. 3D). The C16S form stabilizes three water molecules that interact with each other within the active site (Fig. 3B): Wat1, Wat2, and Wat3. This C16S structure can represent a reduced form. Wat1 in the C16S active site is well superimposed onto the only water molecule (Wat1) in the Sec-to-Cys active site. This water molecule interacts with the side chains of Tyr47 (2.7 Å), Glu55 (2.6 Å), and Tyr90 (2.9 Å), similar to Wat1 in the Sec-to-Cys active site. Wat2 and Wat3 interact with Ser16 (3.1 Å) and Glu55 (2.7 Å), respectively. Interestingly, when the C16S structure was superimposed onto the substrate-bound NmMsrA structure [22], the Wat1, Wat2, and Wat3 were similarly positioned at the sulfoxide oxygen, sulfur, and e methyl group of the substrate, respectively, as in the substrate-bound NmMsrA. In contrast, no water molecules are present within the active site of E55A (Fig. 3C), and an enhanced electron density associated with the Cys16 side chain corresponds to the sulfenic oxygen, similar to the structure of the Sec-to-Cys MsrA. In particular, the Gln89 side chain is re-oriented outward due to the loss of interaction with Glu55.
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Fig. 4. Comparison of the substrate-bound CoMsrA structure (magenta) with the NmMsrA structure (gray). The methionine-S-sulfoxide (Met-S-O)-bound CoMsrA (E55D) structure is superimposed onto the NmMsrA (C51S) structure in a complex with its substrate Ac-Met-S-SO-NHMe (SSM). The interactions of sulfoxide oxygen and sulfur atoms with active site residues are shown as dashed lines in red (CoMsrA) and black (NmMsrA). The figure is displayed in wall-eye stereoview using Pymol.
Fig. 5. Proposed catalytic mechanism for CoMsrA. Glu55 protonates and stabilizes the sulfoxide oxygen (I and II). Glu55 may function for proton transfer from the thiol of catalytic Cys16 to the sulfoxide oxygen via a water molecule. The protonated sulfoxide oxygen is also stabilized by hydrogen-bonding with Tyr90 (III). Nucleophilic attack of the protonated sulfoxide by the thiolate of Cys16 forms the sulfurane intermediate (III). Tyr90 donates a proton to facilitate formation of sulfonium cation (IV). Another water molecule reacts with the sulfonium cation to form the sulfenic acid on Cys16 and release Met as a product (V). The sulfenic acid is reduced to the thiol form by reductants directly or with unknown mechanism(s) (VI).
Fig. 6. Surface models of the active site pocket. The active sites from C16S (A), E55D (B) and Sec-to-Cys (C) are represented by surface models. The active site residues and the substrate methionine-S-sulfoxide (Met-S-O) are displayed in stick models. The sulfenic oxygen of Cys16 in (C) is indicated in red. The surface models display conformational changes in the active site pockets with redox state: reduced (A) to substrate-bound (B) to oxidized (sulfenic acid) (C) forms.
In the E55D variant, the extended electron density brought about by the Cys16 side chain corresponds to the substrate methionine sulfoxide (Fig. 3D and E). The methionine sulfoxide side chain is defined through its electron density, but the main chain appears disordered, increasing the B factor to 49 Å2. The sulfoxide oxygen of the substrate can be stabilized by Gln89 (3.5 Å) and Tyr90 (3.1 Å), and its sulfur atom lies 3.2 Å from that of Cys16. The e methyl group carbon faces towards the hydrophobic site consisting of Phe17 and Trp18 residues. Interestingly, the Tyr47 side chain shows a poor electron density in the E55D active site, in con-
trast to the other three structures (Fig. 3D). It therefore seems that the Tyr47 side chain is disordered from substrate binding. To date, only one substrate-bound structure of MsrA has been reported from N. meningitidis using a catalytic C51S mutant [22]. The orientation of the substrate and its interactions with the active site residues were compared between CoMsrA and NmMsrA (Fig. 4). Notably, the main chain of methionine sulfoxide in the NmMsrA structure is disordered, like the substrate-bound CoMsrA form. In the NmMsrA structure, the sulfoxide oxygen of the substrate is stabilized by the side chains of Tyr82, Glu94, and
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Tyr134. In the CoMsrA, the sulfoxide oxygen is stabilized by Tyr90 (Tyr134 in NmMsrA) and Gln89, but loses its interaction with Asp55 (Glu94 in NmMsrA), due to changes in side chain length from Glu55 to Asp. The interaction of sulfoxide oxygen with Tyr47 (Tyr82 in NmMsrA) is unclear, due to the disordered side chain of this residue in the structure. The sulfur atom of the substrate is located at a similar distance from the catalytic Cys residues in both CoMsrA and NmMsrA structures (3.2 and 3.3 Å, respectively). The e methyl group carbon is located in the hydrophobic site in both structures. Collectively, the substrate binding in the CoMsrA structure agrees with the previously characterized NmMsrA structure. Glu55 is a well-conserved residue in MsrAs, and is essential for catalytic activity via its stabilization of the sulfurane transition state [34,35]. The conserved Glu55 residue acts as a proton donor to the sulfoxide oxygen. Notably, the E55D variant had almost no catalytic activity, despite the presence of catalytic Cys16 residue. This dramatic reduction in activity can be caused by the loss of interaction of Asp55 with the sulfoxide oxygen, thereby preventing proton donation. Collectively, the structural analysis reveals that the length of the Glu55 side chain is an important determinant of the proton donor function of this residue.
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of this protein. The CoMsrA has a unique helical domain in its Cterminal region that interacts with the catalytic domain and is critical for enzyme activity. This domain appears to be conserved in several selenoprotein MsrAs. Our study also sheds light on the architecture of the 1-Cys MsrA active site, including conformational changes depending on redox state, and the roles of active site residues in substrate recognition and catalysis. Acknowledgments We thank the staff of beamlines 4A at the Pohang Accelerator Laboratory and BL1A at the Photon Factory for technical support. We are also grateful to the staff at the Korea Basic Science Institute (Daejeon, Korea) for the use of a mosquito crystallization robot and the Rigaku MicroMax-007HF X-ray generator. This work was supported by grants from the National Research Foundation of Korea (2011-0028166 and 2013-044795). 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.2013.12.024.
Proposed catalytic mechanism of CoMsrA References Collectively, our structural and biochemical analyses suggest a catalytic mechanism for CoMsrA (Fig. 5). Glu55 acts as a proton donor to the sulfoxide oxygen. This residue may function for proton transfer from the thiol of catalytic Cys16 to the sulfoxide oxygen via a water molecule [34]. The protonated sulfoxide is stabilized by hydrogen-bonding with Tyr90 and Glu55. The thiolate of Cys16 then nucleophilically attacks the protonated sulfoxide to form the sulfurane intermediate. Tyr90 acts as the second proton donor to facilitate formation of the sulfonium cation intermediate. Another water molecule reacts with the sulfonium cation intermediate to form the Cys16 sulfenic acid and release Met as a product. The Cys16 sulfenic acid form can be reduced back to the thiol form by reducing agents directly or with unknown mechanism(s). Conformational changes in the active site pocket with redox state As mentioned, the Sec-to-Cys MsrA protein structure represents an oxidized (sulfenic acid) form, the C16S variant structure a reduced form, and the E55D variant structure a substrate-bound form. The overall structures of the three variants are highly similar to the Sec-to-Cys MsrA, with an r.m.s.d. between 0.14 and 0.17 Å. However, the active site pocket appears to change depending on the redox state of the enzyme (Fig. 6). In the reduced form (C16S variant), the active site residues form an ‘open’ active site pocket where the Phe17 residue is located at the bottom. The active pocket has a small size that only binds the terminal side chain of the substrate, including the sulfoxide moiety and e methyl group. This ‘open’ active site pocket appears to remain in a substrate-bound form (E55D variant) with a small change. In the sulfenic acid form (Sec-to-Cys MsrA structure), the sulfenic oxygen of Cys16 is located within the active site pocket, thereby making a ‘closed’ active site to which the substrate cannot bind. On the other hand, the active site pocket of the E55A variant is abnormally open at one side by the change of residue Gln89 (as mentioned above) and thus gets abolished (Supplementary Fig. S4). The data suggest that Glu55 plays an important role in formation of the active site pocket. In summary, we report the first structure of the 1-Cys type selenoprotein form of MsrA, in which Sec was replaced with Cys. The structures obtained in this study represent structural redox states
[1] R.L. Levine, J. Moskovitz, E.R. Stadtman, IUBMB Life 50 (2000) 301–307. [2] E.R. Stadtman, J. Moskovitz, R.L. Levine, Antioxid. Redox Signal. 5 (2003) 577– 582. [3] N. Brot, L. Weissbach, J. Werth, H. Weissbach, Proc. Natl. Acad. Sci. USA 78 (1981) 2155–2158. [4] J. Moskovitz, Biochim. Biophys. Acta 1703 (2005) 213–219. [5] S. Boschi-Muller, A. Gand, G. Branlant, Arch. Biochem. Biophys. 474 (2008) 266–273. [6] G.V. Kryukov, S. Castellano, S.V. Novoselov, A.V. Lobanov, O. Zehtab, R. Guigo, V.N. Gladyshev, Science 300 (2003) 1439–1443. [7] G.V. Kryukov, V.N. Gladyshev, EMBO Rep. 5 (2004) 538–543. [8] M. Rother, A. Resch, R. Wilting, A. Bock, Biofactors 14 (2001) 75–83. [9] H.Y. Kim, Antioxid. Redox Signal. 19 (2013) 958–969. [10] H.Y. Kim, V.N. Gladyshev, Mol. Biol. Cell 15 (2004) 1055–1064. [11] H.Y. Kim, V.N. Gladyshev, PLoS Biol. 3 (2005) e375. [12] H.Y. Kim, D.E. Fomenko, Y.E. Yoon, V.N. Gladyshev, Biochemistry 45 (2006) 13697–13704. [13] H.Y. Kim, Y. Zhang, B.C. Lee, J.R. Kim, V.N. Gladyshev, Proteins 74 (2009) 1008– 1017. [14] J. Moskovitz, J.M. Poston, B.S. Berlett, N.J. Nosworthy, R. Szczepanowski, E.R. Stadtman, J. Biol. Chem. 275 (2000) 14167–14172. [15] S. Boschi-Muller, S. Azza, S. Sanglier-Cianferani, F. Talfournier, A. Van Dorsselear, G. Branlant, J. Biol. Chem. 275 (2000) 35908–35913. [16] J.C. Lim, Z. You, G. Kim, R.L. Levine, Proc. Natl. Acad. Sci. USA 108 (2011) 10472–10477. [17] W.T. Lowther, N. Brot, H. Weissbach, B.W. Matthews, Biochemistry 39 (2000) 13307–13312. [18] S. Boschi-Muller, S. Azza, G. Branlant, Protein Sci. 10 (2001) 2272–2279. [19] A.B. Taylor, D.M. Benglis Jr., S. Dhandayuthapani, P.J. Hart, J. Bacteriol. 185 (2003) 4119–4126. [20] Y.K. Kim, Y.J. Shin, W.H. Lee, H.Y. Kim, K.Y. Hwang, Mol. Microbiol. 72 (2009) 699–709. [21] L. Tarrago, E. Laugier, P. Rey, Mol. Plant 2 (2009) 202–217. [22] F.M. Ranaivoson, M. Antoine, B. Kauffmann, S. Boschi-Muller, A. Aubry, G. Branlant, F. Favier, J. Mol. Biol. 377 (2008) 268–280. [23] F. Tete-Favier, D. Cobessi, S. Boschi-Muller, S. Azza, G. Branlant, A. Aubry, Structure 8 (2000) 1167–1178. [24] N. Rouhier, B. Kauffmann, F. Tete-Favier, P. Palladino, P. Gans, G. Branlant, J.P. Jacquot, S. Boschi-Muller, J. Biol. Chem. 282 (2007) 3367–3378. [25] J.C. Lim, J.M. Gruschus, B. Ghesquiere, G. Kim, G. Piszczek, N. Tjandra, R.L. Levine, J. Biol. Chem. 287 (2012) 25589–25595. [26] Z. Otwinowski, W. Minor, Methods Enzymol. 276 (1997) 307–326. [27] E. Potterton, P. Briggs, M. Turkenburg, E. Dodson, Acta Crystallogr. D: Biol. Crystallogr. 59 (2003) 1131–1137. [28] P. Emsley, K. Cowtan, Acta Crystallogr. D: Biol. Crystallogr. 60 (2004) 2126– 2132. [29] P.D. Adams, P.V. Afonine, G. Bunkoczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, P.H. Zwart, Acta Crystallogr. D: Biol. Crystallogr. 66 (2010) 213–221.
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Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Structural analysis of 1-Cys type selenoprotein methionine sulfoxide reductase A Eun Hye Lee a, Geun-Hee Kwak b, Moon-Jung Kim b, Hwa-Young Kim b,⇑, Kwang Yeon Hwang a,⇑ a b
Division of 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 22 October 2013 and in revised form 23 December 2013 Available online 8 January 2014 Keywords: Methionine sulfoxide reductase MsrA Selenoprotein Selenocysteine Catalysis Clostridium
a b s t r a c t Methionine sulfoxide reductase A (MsrA) reduces free and protein-based methionine-S-sulfoxide to methionine. Structures of 1-Cys MsrAs lacking a resolving Cys, which interacts with catalytic Cys, are unknown. In addition, no structural information on selenocysteine (Sec)-containing MsrA enzymes has been reported. In this work, we determined the crystal structures of 1-Cys type selenoprotein MsrA from Clostridium oremlandii at 1.6–1.8 Å, including the reduced, oxidized (sulfenic acid), and substrate-bound forms. The overall structure of Clostridium MsrA, consisting of ten a-helices and six b-strands, folds into a catalytic domain and a novel helical domain absent from other known MsrA structures. The helical domain, containing five helices, tightly interacts with the catalytic domain, and is likely critical for catalytic activity due to its association with organizing the active site. This helical domain is also conserved in several selenoprotein MsrAs. Our structural analysis reveals that the side chain length of Glu55 is critical for the proton donor function of this residue. Our structures also provide insights into the architecture of the 1-Cys MsrA active site and the roles of active site residues in substrate recognition and catalysis. Ó 2014 Elsevier Inc. All rights reserved.
Introduction The oxidation of methionine to methionine sulfoxide readily occurs due to cellular reactive oxygen species and may induce significant alterations in protein structure and function [1,2]. However, this modification can be reversed by enzymatic reactions [3]. Methionine sulfoxide reductases (Msrs) are the enzymes that catalyze the reduction of methionine sulfoxide in a stereospecific manner [4,5]. MsrA is specific for the S-form of methionine sulfoxide, whereas MsrB only acts on the R-form. Selenoproteins, selenocysteine (Sec)-containing proteins, are found in all three kingdoms of life [6–8]. Selenoprotein forms of Msrs have been identified in many organisms, from bacteria to humans [9]. The Sec residue located at the catalytic sites of selenoprotein Msrs provides higher catalytic activity compared to Sec-to-Cys mutants or Cys-containing homologs [10–13]. Clostridium oremlandii (strain OhILAs), particularly rich in selenoproteins, is a strictly
⇑ Corresponding authors. Address: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, 170 Hyeonchungno, Namgu, Daegu 705-717, Republic of Korea. Fax: +82 53 629 7093 (H.-Y. Kim). Address: Division of 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). E-mail addresses: [email protected] (H.-Y. Kim), [email protected] (K.Y. Hwang). 0003-9861/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.12.024
anaerobic, Gram-positive bacterium that contains the selenoprotein MsrA [13]. This selenoprotein MsrA consists of 209 residues and has a Sec residue within its active site, but contains no Cys residues. This selenoenzyme appears to be among the most efficient MsrA catalysts known [12,13]. The enzymatic activity of the selenoprotein MsrA is 20-fold higher than its Sec-to-Cys form; thus Sec is critical in the catalysis of this selenoenzyme [13]. However, the Sec-to-Cys form also has an MsrA activity comparable to mammalian Cys-containing MsrA [13]. The conserved active site sequence of MsrA is GCFW(G/H) and includes the catalytic Cys [14]. The catalysis of MsrA is based on common sulfenic acid chemistry [15,16]. MsrAs can be divided into three groups based on the resolving Cys, which interacts with catalytic Cys to form an intramolecular disulfide bond: 3-Cys MsrAs, such as the enzymes from Escherichia coli and Bos taurus, have two resolving Cys [15,17,18]; 2-Cys MsrAs, such as the enzymes from Mycobacterium tuberculosis and Streptococcus pneumoniae [19,20], include a single resolving Cys; and 1-Cys MsrAs, such as the MsrA from Synechocystis sp., contain no resolving Cys [21]. The structures of 3-Cys or 2-Cys MsrAs have been well described [17,19,20,22–25], but no structural information on 1-Cys MsrA has been reported yet. Furthermore, the structure of Sec-containing MsrA is unknown. In this work, the crystal structures of 1-Cys type selenoprotein MsrA from C. oremlandii, in which Sec was replaced with Cys, are
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described for the first time. The structures of reduced, oxidized (sulfenic acid), and substrate-bound forms (all representing structural redox states of the enzyme) have been resolved. The overall structure contains a novel helical domain with five helices. This helical domain interacts with the catalytic domain and is essential for catalytic activity. Also, our data provide structural insights into the roles of active site residues in substrate recognition and catalysis, and conformational changes in the active site pocket based on the redox state of the enzyme. Materials and methods
length of 1.0000 Å, and processed using the HKL2000 package [26]. The crystals belonged to the trigonal space group P3221 in a hexagonal setting with unit cell dimensions of a = b = 80.3 Å, c = 66.9 Å, and contained one molecule in an asymmetric unit. The variants C16S, E55A, and E55D were all treated with 10 mM free methionine-(R,S)-sulfoxide at 25 °C for 30 min prior to crystallization. The crystallization conditions for the variants were similar to that for the Sec-to-Cys MsrA. The data were collected on beamline BL1A at the Photon Factory (Tsukuba, Japan) at a wavelength of 1.0000 Å, and processed using the HKL2000 package. A summary of data collection statistics is provided in Table 1.
Cloning and protein purification
Crystal structure determination and refinement
The sequence of the pET-based C. oremlandii MsrA (CoMsrA) was previously described [13]. Sequences encoding the C16S, E55A, and E55D forms were generated by site-directed mutagenesis using pET-CLOS-MsrA/U16C (Sec-to-Cys form) as a template. The sequence lacking the helical domain (residues 1–144) was PCR-amplified and inserted into the NdeI/XhoI sites of pET21b (Novagen). All constructs included a C-terminal His-tag. Plasmids were introduced into E. coli BL21(DE3) star cells, and recombinant cells were grown in LB medium containing 50 lg/ml ampicillin at 37 °C until the OD600 reached 0.6. The heterologous expression of the recombinant protein was induced by the addition of 0.3 mM isopropyl thiogalactoside (IPTG), after which the cultures were incubated at 18 °C for 16 h. Cells were harvested and the cell pellet lysed by sonication in ice-cold 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 4 mM MgCl2, and 5 mM imidazole. After centrifugation at 17,940g for 50 min, the supernatant was loaded onto a HisTrap column equilibrated in the same lysis buffer. An imidazole gradient (10–500 mM) was applied to the protein-bound HisTrap column to elute the protein. The eluate was concentrated to 2 ml by an Amicon (Millipore; Ultracel regenerated cellulose membrane, MWCO 10 K) and then separated by gel filtration chromatography through a Superdex 200 column in 10 mM Tris–HCl (pH 8.0) and 100 mM NaCl. The >98% purity of CoMsrAs was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) analysis, and the protein solution was concentrated to approximately 10 mg/ml for crystallization.
The crystal structure of CoMsrA was determined by molecular replacement using the structure of E. coli MsrA (EcMsrA, PDB ID: 1ff3) as a search model [23] with the software package Molrep implemented in the CCP4i suite [27]. Further model building was performed using the Coot program [28] and refinement was performed with CCP4 refmac5 and PHENIX [29]. The final model was validated using PROCHECK [30] and had an Rcryst of 17.9% and an Rfree of 21.0%. The structures of the variants were determined using the refined CoMsrA structure by molecular replacement and refinements were similarly performed. A summary of statistics related to structure refinement is provided in Table 1.
MsrA assay and kinetic analysis The MsrA reaction mixture (100 ll) contained 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, 20 mM dithiothreitol (DTT), 0.05–0.8 mM dabsyl-methionine-S-sulfoxide, and 1–30 lg purified MsrAs. The reaction was carried out at 37 °C for 30 min, and its product (dabsyl-Met) was detected by high-performance liquid chromatography. Km and kcat values were determined by non-linear regression using Prism 5 software (GraphPad). Crystallization and data collection Due to the low expression level of selenoprotein, the Sec-to-Cys form was used for crystallization. The initial screening of CoMsrA was achieved using the sitting-drop vapor-diffusion method with crystallization conditions (Hampton Research). Crystals suitable for analysis were obtained by the hanging-drop vapor-diffusion method at 20 °C by combining 1.5 ll protein with 1.5 ll precipitation solution. The best crystals were obtained from a reservoir solution consisting of 0.1 M MES (pH 6.5), 30% (w/v) polyethylene glycol monomethyl ether 5000, and 0.2 M ammonium sulfate. Crystals were snap-frozen in liquid nitrogen and subjected to Xray diffraction. A 1.8 Å resolution native data set was collected on beamline 4A at Pohang Light Source (Pohang, Korea) at a wave-
Results and discussion Overall structure of CoMsrA The native CoMsrA carries a Sec residue within its active site. For structure determination, the Sec-to-Cys form of the enzyme was used due to the low expression of the selenoprotein form. The CoMsrA crystal structure was determined at 1.8 Å resolution. One molecule was included in each asymmetric unit, which comprised residues 6–209 and included one additional Leu residue from the His tag (LEHHHHHH) of the recombinant protein. The five N-terminal residues were disordered. The oligomeric state of CoMsrA in solution was also investigated by analytical ultracentrifugation (Supplementary Fig. S1). The result indicated that CoMsrA is a monomeric protein. The monomeric state of CoMsrA is consistent with the fact that other MsrAs characterized so far are monomeric. The overall structure of CoMsrA, comprised of ten a-helices and six b-strands, folded into a catalytic domain (residues 6–144) and an additional helical domain (residues 145–209) (Fig. 1). The catalytic domain consists of a rolled mixed b-sheet, with the exterior side surrounded by five a-helices (a1–a5). The b-sheet is formed by four anti-parallel (b1–b3–b2–b6) and two short parallel (b4– b5) b-strands. The N- and C-terminal sides of the b-sheet are composed of loop regions. The connecting loops (L1 and L2) on the Cterminal side of the b-sheet are slightly longer than those on the opposite side and participate in the formation of the active site. The catalytic Cys16 residue is located on the loop between the b1 strand and the a1 helix. The helical domain consists of five ahelices (a6–a10) that form a four-helix bundle with a short a-helix insertion between the a6 and the a8 helix. Structural comparison of CoMsrA with EcMsrA The EcMsrA is a 3-Cys MsrA that contains two resolving Cys residues (Cys198 and Cys206) in a C-terminal extension. The structure of EcMsrA features extended N-terminal and C-terminal regions surrounding the core domain [23]. The N-terminal region is not essential for catalytic activity, but the C-terminal region provides
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E.H. Lee et al. / Archives of Biochemistry and Biophysics 545 (2014) 1–8 Table 1 Statistics on crystallographic analyses.
a
Sec-to-Cys
C16S
E55A
E55D
PDB ID code
4LWJ
4LWK
4LWL
4LWM
Data collection Space group Asymmetric unit
P3221 Monomer
P32 Dimer
P3221 Monomer
P3221 Monomer
Cell parameters a, b, c (Å) Resolution (Å) Rsym I/rI Completeness (%) Redundancy
80.3, 80.3, 66.9 1.80 (1.83–1.80)a 0.048 (0.221) 32.8 (3.7) 98.0 (97.9) 2.9 (2.3)
81.0, 81.0, 66.7 1.80 (1.83–1.80) 0.060 (0.207) 59.3 (8.2) 99.4 (97.4) 3.3 (2.5)
81.2, 81.2, 66.9 1.60 (1.63–1.60) 0.066 (0.433) 38.3 (2.3) 98.4 (95.2) 3.5 (2.2)
80.8, 80.8, 66.9 1.80 (1.83–1.80) 0.080(0.333) 19.8 (2.2) 97.1 (91.5) 3.0 (1.7)
Refinement No. of reflections Rwork/Rfree (%)
27,101 17.9/21.0
45,156 17.1/20.6
33,211 19.7/23.4
22,944 17.9/22.3
No. of atoms Protein Ligand/ion Water
1641 9 215
3298 18 414
1637 4 155
1639 14 157
B-factors (Å2) Protein Ligand/ion Water
20.569 18.128 25.723
24.616 31.872 34.483
28.708 27.532 37.617
16.381 39.614 24.340
R.m.s deviations Bond lengths (Å) Bond angle (°)
0.007 1.016
0.007 1.003
0.007 1.019
0.015 1.487
Ramachandran plot (%) Most favored Allowed Disallowed
98.5 1.5 0
98.0 2.0 0
98.5 1.5 0
98.0 2.0 0
Values for the highest resolution shell are given in parentheses.
The overall structure of the CoMsrA catalytic domain is similar to EcMsrA, and the catalytic Cys residues, Cys16 in CoMsrA and Cys51 in EcMsrA, coincide (Supplementary Fig. S2). The CoMsrA shares the conserved active site residues with EcMsrA but it shows structural differences near the active site. There are two long loops, L1 (residues 39–53) and L2 (residues 79–92), contributing to formation of the active site. In contrast, EcMsrA contains a helix in the corresponding L1 region and a short b-hairpin in the corresponding L2 region. The short b-hairpin contains an Asp129 residue that contributes to substrate binding by interacting with the backbone nitrogen of the substrate [22]. This residue is conserved in 3-Cys or 2-Cys MsrAs, and the substitution of the Asp129 to Ala leads to a 16-fold increase in Km value [32]. However, the highly conserved Asp residue is absent in CoMsrA. The absence of the conserved Asp in CoMsrA may cause a relatively higher Km value for this enzyme, as characterized previously [13] and in this study (Table 2). Fig. 1. The overall structure of CoMsrA. The structure comprises a 144-residue catalytic domain (pale cyan) and a 65-residue helical domain (red) arranged into 5 helices. The former includes all the catalytic residues. The catalytic C16 residue is represented by a stick model. The specific loop regions L1 and L2 are colored yellow and orange, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the two resolving Cys residues required for thioredoxin-dependent regeneration [18,31]. This flexible C-terminal region, including the resolving Cys residues, is absent from 1-Cys CoMsrA; instead, its catalytic domain is followed by the distinct helical domain (as mentioned above). Other MsrA homologs similar to EcMsrA in structure (e.g., from B. taurus, M. tuberculosis, and Populus trichocarpa) do not include a homolog of the CoMsrA helical domain [17,19,24]. The superposition of the CoMsrA structure on EcMsrA produced a root mean square deviation (r.m.s.d.) of 1.1 Å over 136 Ca atoms.
Interactions between the helical domain and catalytic domain The helical domain has tight interactions with the catalytic domain using the a8 helix located between the catalytic domain a1 and a5 helices (Fig. 2). The hydrophobic stack of Trp18 and Table 2 Kinetic parameters of Sec-to-Cys MsrA and its mutants.
a
1
Form
kcat (min
)
Sec-to-Cys E55A E55D DH-domaina
160 ± 30 0.07 ± 0.015 0.19 ± 0.06 0.26 ± 0.09
Km (mM)
kcat/Km (min
1.92 ± 0.48 0.75 ± 0.28 5.29 ± 1.85 2.54 ± 1.12
83.3 0.09 0.03 0.10
1
DH-domain is a truncated form of MsrA lacking the helical domain.
mM
1
)
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Tyr140 on a1 and a5 helices, respectively, closely holds these helices and contributes to active site formation. Tyr141 on the a5 helix is vertically stacked on these three helices at their converging point. The residues Thr166, Arg170, and Asn172 on the a8 helix contribute to interactions with the a1 and a5 helices. The Od atom of Asn172 forms hydrogen bonds with the amide nitrogen of Tyr141 and Leu142 on the a5 helix. The NH1 and NH2 atoms of Arg170 form hydrogen bonds with the backbone oxygens of Gly25 and Ile27 on the a1 helix and Val30 in the following loop. The Oc atom of Thr166 forms a hydrogen bond with the backbone oxygen of Val31 in the following loop.
Essential role of the helical domain for activity The role of the helical domain was explored by measuring the catalytic activity of a helical domain-truncated form comprising only residues 1–144. In a previous study, an EcMsrA truncated form (containing residues 42–194), which corresponds well to the structure of the helical domain-truncated CoMsrA, showed similar activity to the wild-type in the presence of DTT [18], indicating that the EcMsrA C-terminal region is only required for thioredoxin-dependent regeneration. Due to the high structural similarity of the CoMsrA catalytic domain to the truncated EcMsrA (r.m.s.d. 1.1 Å), the form of CoMsrA deleted in its helical domain was expected to have significant activity in the DTT-dependent condition. However, its catalytic activity was almost completely abolished in the presence of DTT, showing 800-fold lower catalytic efficiency (kcat/Km) compared to the full-length form (Table 2). These data indicate the requirement of the helical domain for catalysis. The helical domain is closely associated with the catalytic domain, interacting with the latter’s a1 and a5 helices via the a8 helix (Fig. 2). Notably, the hydrophobic stack of Trp18 and Tyr140 on the a1 and a5 helices, respectively, contributes to active site formation (Fig. 3A). Thus, the interaction of the helical domain with the catalytic domain may be critical for maintaining active
site organization for catalysis. This interaction might also affect overall folding of the catalytic domain. Further studies will be needed to understand the functional implications of the domain interactions. Conservation of the helical domain in selenoprotein MsrAs To examine the conservation of the helical domain in selenoprotein MsrAs, a multiple sequence alignment was performed (Supplementary Fig. S3). To date, eight selenoprotein MsrA sequences have been reported at UniProt database and may be grouped into two subclasses with respect to the length of the Cterminal region. Two have a short C-terminal tail (10 residues) and are grouped into ‘subclass I’. The other six, including CoMsrA, contain a C-terminal extension (65 residues) and are grouped into ‘subclass II’. The subclass I proteins contain a conserved GUFWH motif and two Cys residues at positions 51 and 88 (numbering based on CoMsrA sequence) that do not serve as a resolving Cys [12]. All subclass II proteins contain the conserved GUFWG motif with no Cys residues in the entire sequence, but one MsrA with two Cys in its C-terminal extension region. These C-terminal extension domains have been predicted to form three to five a-helices by secondary structure prediction using Xtalpred [33]. Overall, the data suggest that the helical domain is conserved in subclass II selenoprotein MsrAs. CoMsrA active site and substrate binding Despite some structural differences around the active site, the architecture of the CoMsrA active site is similar to other MsrA homologs [17,19,23]. A hydrophobic pocket formed by Phe17 and Trp18 in the conserved GCFWG motif occupies one side of the site, while the Tyr47, Glu55, and Tyr90 side chains create a hydrophilic region on the other side (Fig. 3A). Interestingly, the bulge in electron density corresponding to the Cys16 side chain is recognizable
Fig. 2. Interaction of the helical domain with the catalytic domain. Interactions between the helical (red) and catalytic domain (pale cyan) are represented. The helical domain a8 closely interacts with the catalytic domain a1 and a5 through hydrogen bonds. The interacting residues are displayed in a stick model, and hydrogen bonds between the residues are indicated as dashed lines. The two interacting regions are magnified in right squares a and b. (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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Fig. 3. The active site of CoMsrA and its substrate recognition. The active site residues are represented by a stick model, and an electron density map relevant for binding is shown. (A) Active site of the Sec-to-Cys CoMsrA. A water molecule (Wat1, red sphere) is bound at the active site, where it interacts with four residues, including the C16 sulfenic acid form. The bulge of electron density is equivalent to the C16 Od sulfenic oxygen. (B) Active site of the C16S variant. Three water molecules (Wat1, Wat2, and Wat3) are bound at the active site of the C16S variant. (C) Active site of the E55A variant. The Q89 side chain is twisted away from the active site due to loss of the interaction with the E55 side chain. The C16 side chain is present in a sulfenic acid form, similar to Sec-to-Cys. (D) Active site of the substrate-bound E55D variant. The substrate methionine-S-sulfoxide (Met-S-O, gray stick model) is observed at the active site. The electron density associated with the methionine sulfoxide terminal side chain is strong, but the backbone electron density is weak. The electron density for the aromatic ring of Y47 residue is weak. The sulfoxide oxygen atom is stabilized via Q89 and Y90 and the sulfur atom interacts with the C16 sulfur atom. The 2Fo-Fc map contoured at 1.5r shows the electron density for bound molecules at the active site. (E) A stereoview of the active site of substrate-bound E55D. The active site residues (magenta) and substrate (gray) are displayed in the stick model. Met-S-O, methionine-S-sulfoxide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
as the sulfenic acid hydroxyl group. Thus, this structure represents an sulfenic acid form that has also been reported in Neisseria meningitidis MsrA (NmMsrA, PDB ID: 3bqg) [22]. The Od sulfenic oxygen atom of Cys16 lies 3.5 Å from a water molecule (Wat1). Wat1 is stabilized in the active site by its interaction with the Tyr47 (2.7 Å), Glu55 (2.6 Å), and Tyr90 (2.7 Å) side chains. Within the active site, the Gln89 Ne2 atom interacts with the Glu55 Oe2 atom (2.7 Å in distance), while its side chain extends outside the active site in the E. coli and bovine homologs [17,23]. On the opposite side, a highly conserved hydrophobic pocket is formed through a stable p–p interaction between the Trp18 indole ring and the Tyr140 aromatic side chain and by the interaction of the Trp18 indole ring with the His137 side chain. The nature of the CoMsrA active site was further investigated through a comparison of the structures of the three single residue variants (C16S, E55A, and E55D) in the active site (Fig. 3B–E). The C16S variant was catalytically inactive, while the E55A and E55D mutants were almost inactive (Table 2). The kcat values of E55A and E55D mutants were 2300- and 840-fold lower, respectively, than that of Sec-to-Cys MsrA. To obtain a substrate-bound form structure, the variant CoMsrAs were each co-crystallized with the
substrate methionine sulfoxide. However, an electron density map consistent with the substrate was only produced by the E55D variant (Fig. 3D). The C16S form stabilizes three water molecules that interact with each other within the active site (Fig. 3B): Wat1, Wat2, and Wat3. This C16S structure can represent a reduced form. Wat1 in the C16S active site is well superimposed onto the only water molecule (Wat1) in the Sec-to-Cys active site. This water molecule interacts with the side chains of Tyr47 (2.7 Å), Glu55 (2.6 Å), and Tyr90 (2.9 Å), similar to Wat1 in the Sec-to-Cys active site. Wat2 and Wat3 interact with Ser16 (3.1 Å) and Glu55 (2.7 Å), respectively. Interestingly, when the C16S structure was superimposed onto the substrate-bound NmMsrA structure [22], the Wat1, Wat2, and Wat3 were similarly positioned at the sulfoxide oxygen, sulfur, and e methyl group of the substrate, respectively, as in the substrate-bound NmMsrA. In contrast, no water molecules are present within the active site of E55A (Fig. 3C), and an enhanced electron density associated with the Cys16 side chain corresponds to the sulfenic oxygen, similar to the structure of the Sec-to-Cys MsrA. In particular, the Gln89 side chain is re-oriented outward due to the loss of interaction with Glu55.
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Fig. 4. Comparison of the substrate-bound CoMsrA structure (magenta) with the NmMsrA structure (gray). The methionine-S-sulfoxide (Met-S-O)-bound CoMsrA (E55D) structure is superimposed onto the NmMsrA (C51S) structure in a complex with its substrate Ac-Met-S-SO-NHMe (SSM). The interactions of sulfoxide oxygen and sulfur atoms with active site residues are shown as dashed lines in red (CoMsrA) and black (NmMsrA). The figure is displayed in wall-eye stereoview using Pymol.
Fig. 5. Proposed catalytic mechanism for CoMsrA. Glu55 protonates and stabilizes the sulfoxide oxygen (I and II). Glu55 may function for proton transfer from the thiol of catalytic Cys16 to the sulfoxide oxygen via a water molecule. The protonated sulfoxide oxygen is also stabilized by hydrogen-bonding with Tyr90 (III). Nucleophilic attack of the protonated sulfoxide by the thiolate of Cys16 forms the sulfurane intermediate (III). Tyr90 donates a proton to facilitate formation of sulfonium cation (IV). Another water molecule reacts with the sulfonium cation to form the sulfenic acid on Cys16 and release Met as a product (V). The sulfenic acid is reduced to the thiol form by reductants directly or with unknown mechanism(s) (VI).
Fig. 6. Surface models of the active site pocket. The active sites from C16S (A), E55D (B) and Sec-to-Cys (C) are represented by surface models. The active site residues and the substrate methionine-S-sulfoxide (Met-S-O) are displayed in stick models. The sulfenic oxygen of Cys16 in (C) is indicated in red. The surface models display conformational changes in the active site pockets with redox state: reduced (A) to substrate-bound (B) to oxidized (sulfenic acid) (C) forms.
In the E55D variant, the extended electron density brought about by the Cys16 side chain corresponds to the substrate methionine sulfoxide (Fig. 3D and E). The methionine sulfoxide side chain is defined through its electron density, but the main chain appears disordered, increasing the B factor to 49 Å2. The sulfoxide oxygen of the substrate can be stabilized by Gln89 (3.5 Å) and Tyr90 (3.1 Å), and its sulfur atom lies 3.2 Å from that of Cys16. The e methyl group carbon faces towards the hydrophobic site consisting of Phe17 and Trp18 residues. Interestingly, the Tyr47 side chain shows a poor electron density in the E55D active site, in con-
trast to the other three structures (Fig. 3D). It therefore seems that the Tyr47 side chain is disordered from substrate binding. To date, only one substrate-bound structure of MsrA has been reported from N. meningitidis using a catalytic C51S mutant [22]. The orientation of the substrate and its interactions with the active site residues were compared between CoMsrA and NmMsrA (Fig. 4). Notably, the main chain of methionine sulfoxide in the NmMsrA structure is disordered, like the substrate-bound CoMsrA form. In the NmMsrA structure, the sulfoxide oxygen of the substrate is stabilized by the side chains of Tyr82, Glu94, and
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Tyr134. In the CoMsrA, the sulfoxide oxygen is stabilized by Tyr90 (Tyr134 in NmMsrA) and Gln89, but loses its interaction with Asp55 (Glu94 in NmMsrA), due to changes in side chain length from Glu55 to Asp. The interaction of sulfoxide oxygen with Tyr47 (Tyr82 in NmMsrA) is unclear, due to the disordered side chain of this residue in the structure. The sulfur atom of the substrate is located at a similar distance from the catalytic Cys residues in both CoMsrA and NmMsrA structures (3.2 and 3.3 Å, respectively). The e methyl group carbon is located in the hydrophobic site in both structures. Collectively, the substrate binding in the CoMsrA structure agrees with the previously characterized NmMsrA structure. Glu55 is a well-conserved residue in MsrAs, and is essential for catalytic activity via its stabilization of the sulfurane transition state [34,35]. The conserved Glu55 residue acts as a proton donor to the sulfoxide oxygen. Notably, the E55D variant had almost no catalytic activity, despite the presence of catalytic Cys16 residue. This dramatic reduction in activity can be caused by the loss of interaction of Asp55 with the sulfoxide oxygen, thereby preventing proton donation. Collectively, the structural analysis reveals that the length of the Glu55 side chain is an important determinant of the proton donor function of this residue.
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of this protein. The CoMsrA has a unique helical domain in its Cterminal region that interacts with the catalytic domain and is critical for enzyme activity. This domain appears to be conserved in several selenoprotein MsrAs. Our study also sheds light on the architecture of the 1-Cys MsrA active site, including conformational changes depending on redox state, and the roles of active site residues in substrate recognition and catalysis. Acknowledgments We thank the staff of beamlines 4A at the Pohang Accelerator Laboratory and BL1A at the Photon Factory for technical support. We are also grateful to the staff at the Korea Basic Science Institute (Daejeon, Korea) for the use of a mosquito crystallization robot and the Rigaku MicroMax-007HF X-ray generator. This work was supported by grants from the National Research Foundation of Korea (2011-0028166 and 2013-044795). 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.2013.12.024.
Proposed catalytic mechanism of CoMsrA References Collectively, our structural and biochemical analyses suggest a catalytic mechanism for CoMsrA (Fig. 5). Glu55 acts as a proton donor to the sulfoxide oxygen. This residue may function for proton transfer from the thiol of catalytic Cys16 to the sulfoxide oxygen via a water molecule [34]. The protonated sulfoxide is stabilized by hydrogen-bonding with Tyr90 and Glu55. The thiolate of Cys16 then nucleophilically attacks the protonated sulfoxide to form the sulfurane intermediate. Tyr90 acts as the second proton donor to facilitate formation of the sulfonium cation intermediate. Another water molecule reacts with the sulfonium cation intermediate to form the Cys16 sulfenic acid and release Met as a product. The Cys16 sulfenic acid form can be reduced back to the thiol form by reducing agents directly or with unknown mechanism(s). Conformational changes in the active site pocket with redox state As mentioned, the Sec-to-Cys MsrA protein structure represents an oxidized (sulfenic acid) form, the C16S variant structure a reduced form, and the E55D variant structure a substrate-bound form. The overall structures of the three variants are highly similar to the Sec-to-Cys MsrA, with an r.m.s.d. between 0.14 and 0.17 Å. However, the active site pocket appears to change depending on the redox state of the enzyme (Fig. 6). In the reduced form (C16S variant), the active site residues form an ‘open’ active site pocket where the Phe17 residue is located at the bottom. The active pocket has a small size that only binds the terminal side chain of the substrate, including the sulfoxide moiety and e methyl group. This ‘open’ active site pocket appears to remain in a substrate-bound form (E55D variant) with a small change. In the sulfenic acid form (Sec-to-Cys MsrA structure), the sulfenic oxygen of Cys16 is located within the active site pocket, thereby making a ‘closed’ active site to which the substrate cannot bind. On the other hand, the active site pocket of the E55A variant is abnormally open at one side by the change of residue Gln89 (as mentioned above) and thus gets abolished (Supplementary Fig. S4). The data suggest that Glu55 plays an important role in formation of the active site pocket. In summary, we report the first structure of the 1-Cys type selenoprotein form of MsrA, in which Sec was replaced with Cys. The structures obtained in this study represent structural redox states
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