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Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori
Biochimica et Biophysica Acta 1810 (2011) 1355–1360
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori Yoshimitsu Kakuta a, Kazuhiro Usuda b, Takashi Nakashima a, Makoto Kimura a, Yoichi Aso a, Kohji Yamamoto a,⁎ a b
Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
a r t i c l e
i n f o
Article history: Received 2 May 2011 Received in revised form 26 June 2011 Accepted 27 June 2011 Available online 5 July 2011 Keywords: Crystal structure Glutathione Glutathione transferase Lepidoptera Site-directed mutagenesis
a b s t r a c t Background: Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues. Methods: The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of sitedirected mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu. Results: We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features. Conclusions: This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathionebinding region of bmGSTu are critical for catalytic function. General Significance: Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The glutathione transferases (GSTs) [EC 2.5.1.18] are ubiquitously expressed enzymes that are responsible for the cellular detoxification of diverse xenobiotics and endogenous substances through conjugation with reduced glutathione [1,2]. To date, multiple classes of GSTs have been defined, based on differences in amino acid sequence, viz., the Alpha, Mu, Pi, Omega, Sigma, Theta, and Zeta classes in mammals [3], and Delta, Epsilon, Omega, Sigma, Theta, and Zeta classes in dipteran insects such as Anopheles gambiae and Drosophila melanogaster [4]. Insect GSTs are of particular interest, given their role in insecticide metabolism [5–7]. In the case of Lepidoptera, little is known about GSTs other than the Sigma, Omega, Zeta, and Delta-class GSTs of the silkworm
Bombyx mori [8–12] and the Sigma-class GST of the fall webworm Hyphantria cunea [13]. Recently, a novel, unclassified GST was identified in B. mori, which has temporarily been named bmGSTu [14]. In a previous study [14], we found preferential expression of bmGSTu mRNA in the fat body of an insecticide-resistant silkworm strain after exposure to chemical insecticide. Because the silkworm is a model animal for the study of lepidopteran insects, which includes a number of agricultural pests, comprehensive study of silkworm GSTs will be indispensable. In this study, we investigated, using recombinant proteins overexpressed in Escherichia coli, the three-dimensional crystalline structure of bmGSTu and the structure–function relationships in its catalytic action. 2. Materials and methods 2.1. Crystallization and data collection
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; GSH, glutathione; GST, glutathione transferase; GTX, S-hexyl glutathione; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ⁎ Corresponding author at: 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: + 81 92 621 4991; fax: + 81 92 624 1011. E-mail address: [email protected] (K. Yamamoto). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.022
A cDNA encoding bmGSTu, which we had previously identified, was inserted into pET-11b and transformed into BL21 (DE3) cells [14]. Recombinant bmGSTu was purified according to the previously reported method [11] and concentrated to approximately 10 mg/l in
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20 mM Tris–HCl buffer, pH 8.0, containing 0.2 M NaCl. Crystallization was carried out with the sitting-drop vapor diffusion method at 20 °C by using Crystal Screen Kits (Hampton Research), including PEG/Ion-1 and −2, Index, Crystal Screen-1 and −2, and Wizard-1 and-2, as reservoir solutions. Each drop was formed by mixing equal volumes (0.2:0.2 μl) of protein and reservoir solutions. Crystals suitable for Xray analysis were grown for 1 week with 1.6 M sodium citrate. Crystal structure data, derived by synchrotron radiation at beamline BL38B1 in SPring-8 under cryogenic conditions, with crystals soaked in cryoprotectant solution containing 30% (v/v) glycerol, and cooled to 100 K in a nitrogen gas stream, were collected. The diffraction data were processed using the HKL2000 package [15].
2.2. Structure determination The crystal structure of bmGSTu was determined by the molecular replacement method using the program MOLREP [16]. Unless otherwise indicated, the research model used for superposition was agGSTd1-6, a Delta-class GST of A. gambiae (PDB ID: 1PN9). The structure was refined using REFMAC5 [17] with diffraction data for bmGSTu at 2.1 Å resolution. The stereochemical quality of the final model was evaluated by Coot [18], and ROCHECK [19]. Crystallographic parameters and refinement statistics are summarized in Table 1. Figures were prepared using PyMOL (http://pymol.sourceforge.net). The atomic coordinates and structure factors of bmGSTu have been deposited in the Protein Data Bank (PDB ID: 3AY8). Homology alignment of deduced amino acid sequences was performed using ClustalW (ver. 1.83), with 10 and 0.2 as the gap creation penalty and the gap extension, respectively.
2.3. Site-directed mutagenesis and measurements of enzyme activity Mutants of bmGSTu, in which single amino acid residues had been substituted, were constructed using the Quick-Change Site-Directed Mutagenesis Kit (Agilent Technologies, Wilmington, USA) to introduce point mutations in the plasmid harboring bmGSTu wild-type enzyme [14]. The integrity of mutant cDNA constructs were assessed by DNA sequencing. Recombinant GSTs were overexpressed from these constructs and purified as described in Section 2.1 for the wild-type GST. Final protein preparations were subjected to SDS-PAGE using a 15% polyacrylamide slab gel containing 0.1% SDS [20], followed by band-visualization with Coomassie Brilliant Blue R250. Protein concentration was measured using a Protein Assay Kit (Bio-Rad Laboratories) with bovine serum albumin as a standard. The GST activity for wild-type and mutant constructs was assayed as described previously [14,21]. The reaction mixture contained 0.5 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 5 mM glutathione (γGlu-Cys-Gly, GSH) as substrates, dissolved in 50 mM sodium phosphate buffer, pH 6.5. The reaction was monitored by change in absorbance at 340 nm per min, at 30 °C; this was converted into moles CDNB conjugated per minute per milligram of protein by using the molar extinction coefficient of the resultant 2,4-dinitrophenylglutathione (ε340 = 9600 M − 1 cm − 1). Kinetic parameters (Km and Vmax) were obtained from a double-reciprocal plot of data generated by varying the concentrations of CDNB or GSH.
3. Results 3.1. Structural determination and refinement
Table 1 Data collection and refinement statistics. Data collection Space group Unit cell parameters Beam line Wavelength (Å) Resolution range (Å) Number of reflections Observed/Unique Redundancy Rsyma,b I/σ (I)a Completeness (%) Refinement statistics Resolution range (Å) Number of reflections Working set/test set Completeness (%) Rcrystc (%)/Rfreed (%) Root mean square deviations Bond lengths (Å)/bond angles (°) Average B–factor (Å2)/number of atoms Protein Glycerol Water Ramachandran analysis Most favored (%) Allowed (%) Generously allowed (%) Disallowed (%) a
P3221 a = b = 71.40 Å, c = 90.34 Å SPring–8 BL38B1 1.000 50.0–2.10 161,497/15,988 10.0 (10.1) 0.075 (0.447) 49.6 (7.6) 99.9 (100.0)
25.5–2.1 15,171/796 98.4 22.7/18.5 0.010/1.2 34.7/1830 40.0/6 42.6/130 90.7 8.8 0.5 0.0
Values in parentheses are for the highest-resolution shell. Rsym = Σ(I − ‹I›) / Σ‹I›, where I is the intensity measurement for a given reflection and ‹I› is the average intensity for multiple measurements of this reflection. c Rcryst = Σ|Fobs − Fcal|ΣFobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. d Rfree value was calculated for Rcryst, using only an unrefined randomly chosen subset of reflection data (5%). b
A large amount of recombinant bmGSTu protein was prepared using bacterial expression, and the protein was crystallized in space group P3221 with unit cell dimensions of a = b = 71.40 Å, c = 90.34 Å. The final X-ray data and structural refinement statistics are described in Table 1. The refined model had an Rcryst of 22.7% and Rfree of 18.5% for data with a resolution between 25.5 Å and 2.1 Å. It had geometry with root mean square deviations for bond lengths and angles of 0.010 Å and 1.2 Å, respectively. Ramachandran analysis showed that 90.7% of the dihedral angles were found to be in the most favored regions, 8.8% in the allowed regions and only 0.5% in the generously allowed regions. None of the non-glycine residues were in disallowed regions.
3.2. Structure description of bmGSTu On the basis of above data, the crystal structure of bmGSTu was solved by molecular replacement. This B. mori protein had 43.6% sequence identity with the search model agGSTd1-6, a GST from A. gambiae. After comparison with this model, a homodimer structure was obtained for bmGSTu (data not shown), with each subunit having a large cleft, which is open to the active sites. When secondary structural elements, as defined by the DSSP program [22] were employed, we found that each monomer of bmGSTu adopted the canonical GST-fold, including 8 α-helices and 3 β-strands (Fig. 1A and B). The structure possesses 2 distinct domains: an N-terminal domain (residues 1–78) and a C-terminal domain (residues 89–216) (Fig. 1B) connected by a linker (residues 79–88). The former domain contains 3 β-strands, viz., β1 (residues 4–7), β3 (residues 56–59), and β4 (residues 62–65), and 3 α-helices, viz., α1 (residues 12–24), α2 (residues 43–48), and α3 (residues 67–78). The latter domain consists of the α-helices α4 (residues 89–105), α5 (residues 127– 146), α6 (residues 157–172), α7 (residues 180–189), and α8 (residues 195–212).
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A
B
Fig. 1. Primary and tertiary structure alignment/superposition of bmGSTu with agGSTd1-6. (A) Primary structure alignment. For bmGSTu, α-helices and β-strands are indicated in orange and green, respectively. For agGSTd1-6, α-helices and β-strands are underlined and labeled with α and β, respectively, whereas amino acids identical to those in bmGSTu are indicated by asterisks. (B) Tertiary structure superposition. Green and red colors indicate bmGSTu and agGSTd1-6, respectively. N, amino terminal; C, carboxyl terminal. Starting points of α-helices and β-strands are shown by α and β, respectively.
3.3. Active-site residues In agGSTd1-6, 1 molecule of S-hexyl glutathione (GTX) was bound in the active site of each monomer (Fig. 2). The comparison of bmGSTu with agGSTd1-6 indicated that the inhibitor molecule GTX secured tightly inside the active site pocket formed by the residues Ser12, Gln52, His53, Val55, Glu67, Ser68, Arg69, Tyr108, and Phe116 of bmGSTu; these were superimposed on the corresponding residues Ser9, His50, His38, Ile52, Glu64, Ser65, Arg66, Tyr105, and Tyr113 of agGSTd1-6 (Fig. 2). The active site can be divided into 2 subsites, the GSH-binding site (G-site) and the hydrophilic-binding site (H-site). The G-site of bmGSTu mainly consists of hydrophobic amino acid residues. The GSH moiety of the inhibitor GTX would lie at this site, with its γ-glutamyl region forming hydrogen bonds with the side chain of Glu67 (Glu64 in agGST1-6), the hydroxyl group of Ser68 (Ser65), and the side chain of Arg69 (Arg66) (Fig. 3A). The cysteinyl moiety of GTX appeared to form hydrogen bonds with the main-chain carbonyl of Val55 (Ile52 in agGST1-6) and with the hydroxyl group of Ser12 (Ser9 in agGST1-6) (Fig. 3B). The glycyl portion of GTX, interacting with the side chain of
Gln52 (His50 in agGST1-6), could be in close contact with the side chain of His53 (His38 in agGST1-6) through a hydrogen bond bridged by a water molecule (Fig. 3C). To establish whether the H-site (harboring Tyr108, Arg112, and Phe116) of bmGSTu could bind a substrate, we applied superposition of the structural model with another search model, viz., Mu-class GST from humans (hGSTM1A-1A, PDB ID: 1XWK). From this data, we suggest that these 3 H-site-residues of bmGSTu could interact with a complex of GSH and 2,4-dinitrobenzene (Fig. 4). To assess whether the residues in the proposed bmGSTu active site are in fact involved in catalytic activity, we changed some of the residues to Ala by site-directed mutagenesis. Each of the resulting mutants, namely, H53A, V55A, E67A, S68A, and R69A were purified from E. coli cells and yielded a single band on SDS-PAGE. These 5 mutants were shown to have enzyme activity, and their kinetic parameters for CDNB and GSH were compared with those of the wildtype enzyme (Table 2). With respect to CDNB, the kcat/Km values of H53A and R69A were slightly decreased, whereas those of V55A, E67A, and S68A were extensively decreased. The parameters of R69A were highly similar to those of the wild-type bmGSTu. Similarly, no
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A
Ser9
Fig. 2. Tertiary structure superposition of bmGSTu with agGSTd1-6, showing amino acid residues near the active center. Displays for bmGSTu are indicated in green, and those for agGSTd1-6, in orange, except for the regions of oxygen (red), nitrogen (blue), and sulfur (yellow). GTX is shown in blue. Amino acid residues for bmGSTu and agGSTd1-6 are indicated in black and orange font, respectively.
B
large alteration in kcat/Km for GSH was observed for H53A and R69A. However, these values were decreased for V55A, E67A, and S68A but were slightly increased for R69A. Of the residues tested, Val55 exhibited the most marked changes in kcat/Km after site-directed mutagenesis, followed by Glu67. 4. Discussion In this study, we elucidated the tertiary structure of a novel silkworm GST, bmGSTu. The overall features of bmGSTu, a dimeric protein with a globular shape, were similar to those of other known insect Delta-class GSTs. In the N-terminal domain of other GSTs, which contained 3 α-helices and 4 β-strands that formed a βαβαββα motif, the β3 and β4 strands were antiparallel and were connected by the α1 helix (Fig. 1A and B). Similarly, in bmGSTu, the β3 and β4 strands were antiparallel. However, comparison with agGSTd1-6 revealed that the β2 part was not present in bmGSTu. The lock-and-key motif is crucial for stabilizing hydrophobic interactions of the GST monomers [23,24]. Recently, an insect-specific motif was proposed based on studies on Anopheles dirus GST [25]. The “key” residues Gln65, Arg67, and Phe104 of 1 subunit interact with a hydrophobic area formed by “lock” residues Ala68, Leu103, Phe104, and Val07 of the other subunit in A. dirus Delta-class GST [25]. Our data show the presence of corresponding key residues (Gln67, Arg69, and Tyr108) and lock residues (Ala69, Leu107, Tyr108, and Val09) in bmGSTu, again suggesting similarity of this B. mori enzyme to the insect Delta-class GST. The superposition of the backbones of bmGSTu and agGSTd1-6 revealed that both consist of an N-terminal domain, providing the Gsite and a C-terminal domain, which includes the H-site. The secondary structures of these GSTs were comparable, especially at the G-site, thus providing a starting model for GSH binding in bmGSTu. Thus, it appears that the side-chain glutamyl residue in GSH is stabilized by hydrogen-bond interactions, wherein Val55 adopts a conformation to improve the interactions. GSH is further stabilized by hydrogen bonds between the glycyl residue of GSH and the active-site residues; this interaction is enforced by water-mediated hydrogen
C
Fig. 3. Tertiary structure superposition of bmGSTu with agGSTd1-6, showing amino acid residues near the G-site. Coloring schemes for displays, letters, and GTX are as described in the caption for Fig. 2. Hydrogen bonds are indicated by broken lines. (A) Residues interacting with γ-glutamyl region of GTX. (B) Residues interacting with cysteinyl moiety of GTX. (C) Residues interacting with glycyl portion of GTX. Water molecule is shown by red asterisk.
bonds. Previously, we found that Ser12 in bmGSTu affected the bmGSTu activity after site-directed mutagenesis [14, cf. also Table 2]. In the crystalline structure, the hydroxyl group of Ser12 forms a
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Tyr108 Leu12 His107
Tyr6
Met108
Arg112 GS-DNB Phe116 Tyr115 Fig. 4. Structural features at the H-site of bmGSTu. Tertiary structure superposition of bmGSTu with hGSTM1A-1A. Residues of bmGSTu and hGSTM1A-1A are shown in green and blue, respectively, except for the regions of oxygen (orange), nitrogen (blue), and sulfur (yellow). The substrate indicated in red is the complex of GSH and 2,4dinitrobenzene abbreviated as GS-DNB. Names of amino acid residues for bmGSTu and hGSTM1A-1A are indicated by black and orange font, respectively.
hydrogen bond with the sulfur atom of GSX (Figs. 2 and 3B). A similar result has been obtained for GST from Glycine max, where the cysteine residue of GSH points towards α-helix H2 and away from the catalytic Ser13 [26]. It is possible that the SH group is ionized and acts as a nucleophile in the catalytic reaction. In the crystal structure of the A. dirus Delta-class GST, 5 residues in the G-site (Ile52, Glu64, Ser65, Arg66, and Met101) were shown to interact with GSH [33]. We found that bmGSTu share many similarities with this class of GST. First, bmGSTu had a GSHconjugation activity toward CDNB, as do Delta-class GSTs; second, we have previously found that Tyr7 and Ser12 markedly affected bmGSTu activity [14], which is similar to Delta-, Sigma-, Theta-, and Zeta-class GSTs [28–32]. Therefore, the amino acids corresponding to the 5 GSH-interacting residues were targeted by site-directed mutagenesis. Among the resulting mutants, V55A, as well as E67A and S68A, showed notably reduced catalytic efficiency for CDNB and GSH; however, H53A and R69A were not significantly affected. These results suggest that, in bmGSTu, at least the residues Val55, Glu67, and Ser68, in addition to the previously identified Tyr7 and Ser12 [14], play important roles in catalysis.
Table 2 Comparison of kinetic data. CDNB
GSH
Enzyme Wild type S12A1 H53A V55A E67A S68A R69A
1
Km
Vmax
kcat/Km Km
0.39 (0.06) 0.70 (0.05) 0.30 (0.04) 1.50 (0.06) 1.09 (0.03) N.D.2 0.44 (0.07)
17.7 (0.01) 2.3 (0.23) 14.9 (0.05) 6.6 (0.58) 2.8 (0.37) N.D. 15.4 (0.84)
138.0 20.4 82.7 5.2 10.4 N.D. 102.0
3.94 3.14 0.84 1.34 0.69 2.55 1.03
Vmax (0.61) (0.42) (0.05) (0.13) (0.07) (0.25) (0.07)
30.5 2.45 11.5 2.34 1.21 4.45 12.9
kcat/Km (0.16) (0.21) (0.35) (0.37) (0.07) (0.38) (0.63)
23.7 4.9 22.9 2.1 7.1 17.8 36.4
Kinetic data were determined for CDNB and GSH. Values except those of kcat/Km are expressed as mean (SD) of three independent experiments. Km, Vmax and kcat/Km are expressed as mM, μmol ·mg− 1·min− 1 and min− 1·mM− 1, respectively. 1 The values were obtained in a previous report [14]. 2 N.D.: not determined. The Km value for CDNB (S68A mutant) is above the solubility limit.
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Amongst GSTs, variations in H-site structures are responsible for different substrate specificities. Although the secondary structure of the C-terminal region of bmGSTu is similar to that of agGSTd1-6, the component amino acids are different. The H-site of agGSTd1-6 contains residues that are mostly hydrophobic: Leu6, Ala10, Pro11, Leu33, Met34, Tyr105, Phe108, Tyr113, Ile116, Phe117, Phe203, and Phe207 [27]. In the sequence of bmGSTu, we found that, 5 of the 12 residues, viz., Pro14, Leu36, Tyr108, Phe120, and Phe206, are identical to those in agGSTd1-6, and the remainder, viz., Phe8, Gly13, Phe37, Ile111, Phe116, Leu119, and Val206 are similar to the above agGST1-6 residues. In summary, we described a high-resolution (2.1 Å) crystal structure of bmGSTu, which was found to have similarities with other GSTs. By comparison with a GST of A. gambiae, potential binding sites for GSH and substrates were allocated. We also characterized the amino acid residues of bmGSTu related to catalytic efficiency by employing site-directed mutagenesis. We are currently pursuing cocrystallization of bmGSTu with a suitable insecticide-GSH conjugate to gain further understanding of the molecular basis for detoxification of xenobiotics by this novel GST.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI; 21780049) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mishima Kaiun Memorial Foundation. This work was also supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University.
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[26] L. Chen, P.R. Hall, X.E. Zhaou, H. Ranson, J. Hemingway, E.J. Meehan, Structure of an insect δ-class glutathione S-transferase from a DDT-resistant strain of the malaria vector Anopheles gambiae, Acta Crystallogr. D59 (2003) 2211–2217. [27] I. Axarli, P. Dhavala, A.C. Parageorgiou, N.E. Labrou, Crystal structure of Glycine max glutathione transferase in complex with glutathione: investigation of the mechanism operating by the Tau class glutathione transferases, Biochem. J. 422 (2009) 247–256. [28] D. Sheehan, G. Meade, V.M. Foley, C.A. Dowd, Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily, Biochem. J. 360 (2001) 1–16. [29] A. Gustafsson, P.L. Pettersson, L. Grehn, P. Jemth, B. Mannervik, Role of the glutamyl α-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-1, Biochemistry 40 (2001) 15835–15845. [30] J. Rossjohn, W.J. Mckinstry, A.J. Oakley, D. Verger, J. Flanagan, G. Chelvanayagam, K.L. Tan, P.G. Board, M.W. Parker, Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site, Structure 6 (1998) 309–322. [31] K.L. Tan, G. Chelvanayagam, M.W. Parker, P.G. Board, Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues, Biochem. J. 319 (1996) 315–321. [32] P. Winayanuwattikun, A.J. Ketterman, An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes, J. Biol. Chem. 280 (2005) 31776–31782. [33] P. Winayanuwattikun, A.J. Ketterman, Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase, Biochem. J. 382 (2004) 751–757.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori Yoshimitsu Kakuta a, Kazuhiro Usuda b, Takashi Nakashima a, Makoto Kimura a, Yoichi Aso a, Kohji Yamamoto a,⁎ a b
Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
a r t i c l e
i n f o
Article history: Received 2 May 2011 Received in revised form 26 June 2011 Accepted 27 June 2011 Available online 5 July 2011 Keywords: Crystal structure Glutathione Glutathione transferase Lepidoptera Site-directed mutagenesis
a b s t r a c t Background: Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues. Methods: The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of sitedirected mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu. Results: We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features. Conclusions: This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathionebinding region of bmGSTu are critical for catalytic function. General Significance: Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The glutathione transferases (GSTs) [EC 2.5.1.18] are ubiquitously expressed enzymes that are responsible for the cellular detoxification of diverse xenobiotics and endogenous substances through conjugation with reduced glutathione [1,2]. To date, multiple classes of GSTs have been defined, based on differences in amino acid sequence, viz., the Alpha, Mu, Pi, Omega, Sigma, Theta, and Zeta classes in mammals [3], and Delta, Epsilon, Omega, Sigma, Theta, and Zeta classes in dipteran insects such as Anopheles gambiae and Drosophila melanogaster [4]. Insect GSTs are of particular interest, given their role in insecticide metabolism [5–7]. In the case of Lepidoptera, little is known about GSTs other than the Sigma, Omega, Zeta, and Delta-class GSTs of the silkworm
Bombyx mori [8–12] and the Sigma-class GST of the fall webworm Hyphantria cunea [13]. Recently, a novel, unclassified GST was identified in B. mori, which has temporarily been named bmGSTu [14]. In a previous study [14], we found preferential expression of bmGSTu mRNA in the fat body of an insecticide-resistant silkworm strain after exposure to chemical insecticide. Because the silkworm is a model animal for the study of lepidopteran insects, which includes a number of agricultural pests, comprehensive study of silkworm GSTs will be indispensable. In this study, we investigated, using recombinant proteins overexpressed in Escherichia coli, the three-dimensional crystalline structure of bmGSTu and the structure–function relationships in its catalytic action. 2. Materials and methods 2.1. Crystallization and data collection
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; GSH, glutathione; GST, glutathione transferase; GTX, S-hexyl glutathione; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ⁎ Corresponding author at: 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: + 81 92 621 4991; fax: + 81 92 624 1011. E-mail address: [email protected] (K. Yamamoto). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.022
A cDNA encoding bmGSTu, which we had previously identified, was inserted into pET-11b and transformed into BL21 (DE3) cells [14]. Recombinant bmGSTu was purified according to the previously reported method [11] and concentrated to approximately 10 mg/l in
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20 mM Tris–HCl buffer, pH 8.0, containing 0.2 M NaCl. Crystallization was carried out with the sitting-drop vapor diffusion method at 20 °C by using Crystal Screen Kits (Hampton Research), including PEG/Ion-1 and −2, Index, Crystal Screen-1 and −2, and Wizard-1 and-2, as reservoir solutions. Each drop was formed by mixing equal volumes (0.2:0.2 μl) of protein and reservoir solutions. Crystals suitable for Xray analysis were grown for 1 week with 1.6 M sodium citrate. Crystal structure data, derived by synchrotron radiation at beamline BL38B1 in SPring-8 under cryogenic conditions, with crystals soaked in cryoprotectant solution containing 30% (v/v) glycerol, and cooled to 100 K in a nitrogen gas stream, were collected. The diffraction data were processed using the HKL2000 package [15].
2.2. Structure determination The crystal structure of bmGSTu was determined by the molecular replacement method using the program MOLREP [16]. Unless otherwise indicated, the research model used for superposition was agGSTd1-6, a Delta-class GST of A. gambiae (PDB ID: 1PN9). The structure was refined using REFMAC5 [17] with diffraction data for bmGSTu at 2.1 Å resolution. The stereochemical quality of the final model was evaluated by Coot [18], and ROCHECK [19]. Crystallographic parameters and refinement statistics are summarized in Table 1. Figures were prepared using PyMOL (http://pymol.sourceforge.net). The atomic coordinates and structure factors of bmGSTu have been deposited in the Protein Data Bank (PDB ID: 3AY8). Homology alignment of deduced amino acid sequences was performed using ClustalW (ver. 1.83), with 10 and 0.2 as the gap creation penalty and the gap extension, respectively.
2.3. Site-directed mutagenesis and measurements of enzyme activity Mutants of bmGSTu, in which single amino acid residues had been substituted, were constructed using the Quick-Change Site-Directed Mutagenesis Kit (Agilent Technologies, Wilmington, USA) to introduce point mutations in the plasmid harboring bmGSTu wild-type enzyme [14]. The integrity of mutant cDNA constructs were assessed by DNA sequencing. Recombinant GSTs were overexpressed from these constructs and purified as described in Section 2.1 for the wild-type GST. Final protein preparations were subjected to SDS-PAGE using a 15% polyacrylamide slab gel containing 0.1% SDS [20], followed by band-visualization with Coomassie Brilliant Blue R250. Protein concentration was measured using a Protein Assay Kit (Bio-Rad Laboratories) with bovine serum albumin as a standard. The GST activity for wild-type and mutant constructs was assayed as described previously [14,21]. The reaction mixture contained 0.5 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 5 mM glutathione (γGlu-Cys-Gly, GSH) as substrates, dissolved in 50 mM sodium phosphate buffer, pH 6.5. The reaction was monitored by change in absorbance at 340 nm per min, at 30 °C; this was converted into moles CDNB conjugated per minute per milligram of protein by using the molar extinction coefficient of the resultant 2,4-dinitrophenylglutathione (ε340 = 9600 M − 1 cm − 1). Kinetic parameters (Km and Vmax) were obtained from a double-reciprocal plot of data generated by varying the concentrations of CDNB or GSH.
3. Results 3.1. Structural determination and refinement
Table 1 Data collection and refinement statistics. Data collection Space group Unit cell parameters Beam line Wavelength (Å) Resolution range (Å) Number of reflections Observed/Unique Redundancy Rsyma,b I/σ (I)a Completeness (%) Refinement statistics Resolution range (Å) Number of reflections Working set/test set Completeness (%) Rcrystc (%)/Rfreed (%) Root mean square deviations Bond lengths (Å)/bond angles (°) Average B–factor (Å2)/number of atoms Protein Glycerol Water Ramachandran analysis Most favored (%) Allowed (%) Generously allowed (%) Disallowed (%) a
P3221 a = b = 71.40 Å, c = 90.34 Å SPring–8 BL38B1 1.000 50.0–2.10 161,497/15,988 10.0 (10.1) 0.075 (0.447) 49.6 (7.6) 99.9 (100.0)
25.5–2.1 15,171/796 98.4 22.7/18.5 0.010/1.2 34.7/1830 40.0/6 42.6/130 90.7 8.8 0.5 0.0
Values in parentheses are for the highest-resolution shell. Rsym = Σ(I − ‹I›) / Σ‹I›, where I is the intensity measurement for a given reflection and ‹I› is the average intensity for multiple measurements of this reflection. c Rcryst = Σ|Fobs − Fcal|ΣFobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. d Rfree value was calculated for Rcryst, using only an unrefined randomly chosen subset of reflection data (5%). b
A large amount of recombinant bmGSTu protein was prepared using bacterial expression, and the protein was crystallized in space group P3221 with unit cell dimensions of a = b = 71.40 Å, c = 90.34 Å. The final X-ray data and structural refinement statistics are described in Table 1. The refined model had an Rcryst of 22.7% and Rfree of 18.5% for data with a resolution between 25.5 Å and 2.1 Å. It had geometry with root mean square deviations for bond lengths and angles of 0.010 Å and 1.2 Å, respectively. Ramachandran analysis showed that 90.7% of the dihedral angles were found to be in the most favored regions, 8.8% in the allowed regions and only 0.5% in the generously allowed regions. None of the non-glycine residues were in disallowed regions.
3.2. Structure description of bmGSTu On the basis of above data, the crystal structure of bmGSTu was solved by molecular replacement. This B. mori protein had 43.6% sequence identity with the search model agGSTd1-6, a GST from A. gambiae. After comparison with this model, a homodimer structure was obtained for bmGSTu (data not shown), with each subunit having a large cleft, which is open to the active sites. When secondary structural elements, as defined by the DSSP program [22] were employed, we found that each monomer of bmGSTu adopted the canonical GST-fold, including 8 α-helices and 3 β-strands (Fig. 1A and B). The structure possesses 2 distinct domains: an N-terminal domain (residues 1–78) and a C-terminal domain (residues 89–216) (Fig. 1B) connected by a linker (residues 79–88). The former domain contains 3 β-strands, viz., β1 (residues 4–7), β3 (residues 56–59), and β4 (residues 62–65), and 3 α-helices, viz., α1 (residues 12–24), α2 (residues 43–48), and α3 (residues 67–78). The latter domain consists of the α-helices α4 (residues 89–105), α5 (residues 127– 146), α6 (residues 157–172), α7 (residues 180–189), and α8 (residues 195–212).
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A
B
Fig. 1. Primary and tertiary structure alignment/superposition of bmGSTu with agGSTd1-6. (A) Primary structure alignment. For bmGSTu, α-helices and β-strands are indicated in orange and green, respectively. For agGSTd1-6, α-helices and β-strands are underlined and labeled with α and β, respectively, whereas amino acids identical to those in bmGSTu are indicated by asterisks. (B) Tertiary structure superposition. Green and red colors indicate bmGSTu and agGSTd1-6, respectively. N, amino terminal; C, carboxyl terminal. Starting points of α-helices and β-strands are shown by α and β, respectively.
3.3. Active-site residues In agGSTd1-6, 1 molecule of S-hexyl glutathione (GTX) was bound in the active site of each monomer (Fig. 2). The comparison of bmGSTu with agGSTd1-6 indicated that the inhibitor molecule GTX secured tightly inside the active site pocket formed by the residues Ser12, Gln52, His53, Val55, Glu67, Ser68, Arg69, Tyr108, and Phe116 of bmGSTu; these were superimposed on the corresponding residues Ser9, His50, His38, Ile52, Glu64, Ser65, Arg66, Tyr105, and Tyr113 of agGSTd1-6 (Fig. 2). The active site can be divided into 2 subsites, the GSH-binding site (G-site) and the hydrophilic-binding site (H-site). The G-site of bmGSTu mainly consists of hydrophobic amino acid residues. The GSH moiety of the inhibitor GTX would lie at this site, with its γ-glutamyl region forming hydrogen bonds with the side chain of Glu67 (Glu64 in agGST1-6), the hydroxyl group of Ser68 (Ser65), and the side chain of Arg69 (Arg66) (Fig. 3A). The cysteinyl moiety of GTX appeared to form hydrogen bonds with the main-chain carbonyl of Val55 (Ile52 in agGST1-6) and with the hydroxyl group of Ser12 (Ser9 in agGST1-6) (Fig. 3B). The glycyl portion of GTX, interacting with the side chain of
Gln52 (His50 in agGST1-6), could be in close contact with the side chain of His53 (His38 in agGST1-6) through a hydrogen bond bridged by a water molecule (Fig. 3C). To establish whether the H-site (harboring Tyr108, Arg112, and Phe116) of bmGSTu could bind a substrate, we applied superposition of the structural model with another search model, viz., Mu-class GST from humans (hGSTM1A-1A, PDB ID: 1XWK). From this data, we suggest that these 3 H-site-residues of bmGSTu could interact with a complex of GSH and 2,4-dinitrobenzene (Fig. 4). To assess whether the residues in the proposed bmGSTu active site are in fact involved in catalytic activity, we changed some of the residues to Ala by site-directed mutagenesis. Each of the resulting mutants, namely, H53A, V55A, E67A, S68A, and R69A were purified from E. coli cells and yielded a single band on SDS-PAGE. These 5 mutants were shown to have enzyme activity, and their kinetic parameters for CDNB and GSH were compared with those of the wildtype enzyme (Table 2). With respect to CDNB, the kcat/Km values of H53A and R69A were slightly decreased, whereas those of V55A, E67A, and S68A were extensively decreased. The parameters of R69A were highly similar to those of the wild-type bmGSTu. Similarly, no
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A
Ser9
Fig. 2. Tertiary structure superposition of bmGSTu with agGSTd1-6, showing amino acid residues near the active center. Displays for bmGSTu are indicated in green, and those for agGSTd1-6, in orange, except for the regions of oxygen (red), nitrogen (blue), and sulfur (yellow). GTX is shown in blue. Amino acid residues for bmGSTu and agGSTd1-6 are indicated in black and orange font, respectively.
B
large alteration in kcat/Km for GSH was observed for H53A and R69A. However, these values were decreased for V55A, E67A, and S68A but were slightly increased for R69A. Of the residues tested, Val55 exhibited the most marked changes in kcat/Km after site-directed mutagenesis, followed by Glu67. 4. Discussion In this study, we elucidated the tertiary structure of a novel silkworm GST, bmGSTu. The overall features of bmGSTu, a dimeric protein with a globular shape, were similar to those of other known insect Delta-class GSTs. In the N-terminal domain of other GSTs, which contained 3 α-helices and 4 β-strands that formed a βαβαββα motif, the β3 and β4 strands were antiparallel and were connected by the α1 helix (Fig. 1A and B). Similarly, in bmGSTu, the β3 and β4 strands were antiparallel. However, comparison with agGSTd1-6 revealed that the β2 part was not present in bmGSTu. The lock-and-key motif is crucial for stabilizing hydrophobic interactions of the GST monomers [23,24]. Recently, an insect-specific motif was proposed based on studies on Anopheles dirus GST [25]. The “key” residues Gln65, Arg67, and Phe104 of 1 subunit interact with a hydrophobic area formed by “lock” residues Ala68, Leu103, Phe104, and Val07 of the other subunit in A. dirus Delta-class GST [25]. Our data show the presence of corresponding key residues (Gln67, Arg69, and Tyr108) and lock residues (Ala69, Leu107, Tyr108, and Val09) in bmGSTu, again suggesting similarity of this B. mori enzyme to the insect Delta-class GST. The superposition of the backbones of bmGSTu and agGSTd1-6 revealed that both consist of an N-terminal domain, providing the Gsite and a C-terminal domain, which includes the H-site. The secondary structures of these GSTs were comparable, especially at the G-site, thus providing a starting model for GSH binding in bmGSTu. Thus, it appears that the side-chain glutamyl residue in GSH is stabilized by hydrogen-bond interactions, wherein Val55 adopts a conformation to improve the interactions. GSH is further stabilized by hydrogen bonds between the glycyl residue of GSH and the active-site residues; this interaction is enforced by water-mediated hydrogen
C
Fig. 3. Tertiary structure superposition of bmGSTu with agGSTd1-6, showing amino acid residues near the G-site. Coloring schemes for displays, letters, and GTX are as described in the caption for Fig. 2. Hydrogen bonds are indicated by broken lines. (A) Residues interacting with γ-glutamyl region of GTX. (B) Residues interacting with cysteinyl moiety of GTX. (C) Residues interacting with glycyl portion of GTX. Water molecule is shown by red asterisk.
bonds. Previously, we found that Ser12 in bmGSTu affected the bmGSTu activity after site-directed mutagenesis [14, cf. also Table 2]. In the crystalline structure, the hydroxyl group of Ser12 forms a
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Tyr108 Leu12 His107
Tyr6
Met108
Arg112 GS-DNB Phe116 Tyr115 Fig. 4. Structural features at the H-site of bmGSTu. Tertiary structure superposition of bmGSTu with hGSTM1A-1A. Residues of bmGSTu and hGSTM1A-1A are shown in green and blue, respectively, except for the regions of oxygen (orange), nitrogen (blue), and sulfur (yellow). The substrate indicated in red is the complex of GSH and 2,4dinitrobenzene abbreviated as GS-DNB. Names of amino acid residues for bmGSTu and hGSTM1A-1A are indicated by black and orange font, respectively.
hydrogen bond with the sulfur atom of GSX (Figs. 2 and 3B). A similar result has been obtained for GST from Glycine max, where the cysteine residue of GSH points towards α-helix H2 and away from the catalytic Ser13 [26]. It is possible that the SH group is ionized and acts as a nucleophile in the catalytic reaction. In the crystal structure of the A. dirus Delta-class GST, 5 residues in the G-site (Ile52, Glu64, Ser65, Arg66, and Met101) were shown to interact with GSH [33]. We found that bmGSTu share many similarities with this class of GST. First, bmGSTu had a GSHconjugation activity toward CDNB, as do Delta-class GSTs; second, we have previously found that Tyr7 and Ser12 markedly affected bmGSTu activity [14], which is similar to Delta-, Sigma-, Theta-, and Zeta-class GSTs [28–32]. Therefore, the amino acids corresponding to the 5 GSH-interacting residues were targeted by site-directed mutagenesis. Among the resulting mutants, V55A, as well as E67A and S68A, showed notably reduced catalytic efficiency for CDNB and GSH; however, H53A and R69A were not significantly affected. These results suggest that, in bmGSTu, at least the residues Val55, Glu67, and Ser68, in addition to the previously identified Tyr7 and Ser12 [14], play important roles in catalysis.
Table 2 Comparison of kinetic data. CDNB
GSH
Enzyme Wild type S12A1 H53A V55A E67A S68A R69A
1
Km
Vmax
kcat/Km Km
0.39 (0.06) 0.70 (0.05) 0.30 (0.04) 1.50 (0.06) 1.09 (0.03) N.D.2 0.44 (0.07)
17.7 (0.01) 2.3 (0.23) 14.9 (0.05) 6.6 (0.58) 2.8 (0.37) N.D. 15.4 (0.84)
138.0 20.4 82.7 5.2 10.4 N.D. 102.0
3.94 3.14 0.84 1.34 0.69 2.55 1.03
Vmax (0.61) (0.42) (0.05) (0.13) (0.07) (0.25) (0.07)
30.5 2.45 11.5 2.34 1.21 4.45 12.9
kcat/Km (0.16) (0.21) (0.35) (0.37) (0.07) (0.38) (0.63)
23.7 4.9 22.9 2.1 7.1 17.8 36.4
Kinetic data were determined for CDNB and GSH. Values except those of kcat/Km are expressed as mean (SD) of three independent experiments. Km, Vmax and kcat/Km are expressed as mM, μmol ·mg− 1·min− 1 and min− 1·mM− 1, respectively. 1 The values were obtained in a previous report [14]. 2 N.D.: not determined. The Km value for CDNB (S68A mutant) is above the solubility limit.
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Amongst GSTs, variations in H-site structures are responsible for different substrate specificities. Although the secondary structure of the C-terminal region of bmGSTu is similar to that of agGSTd1-6, the component amino acids are different. The H-site of agGSTd1-6 contains residues that are mostly hydrophobic: Leu6, Ala10, Pro11, Leu33, Met34, Tyr105, Phe108, Tyr113, Ile116, Phe117, Phe203, and Phe207 [27]. In the sequence of bmGSTu, we found that, 5 of the 12 residues, viz., Pro14, Leu36, Tyr108, Phe120, and Phe206, are identical to those in agGSTd1-6, and the remainder, viz., Phe8, Gly13, Phe37, Ile111, Phe116, Leu119, and Val206 are similar to the above agGST1-6 residues. In summary, we described a high-resolution (2.1 Å) crystal structure of bmGSTu, which was found to have similarities with other GSTs. By comparison with a GST of A. gambiae, potential binding sites for GSH and substrates were allocated. We also characterized the amino acid residues of bmGSTu related to catalytic efficiency by employing site-directed mutagenesis. We are currently pursuing cocrystallization of bmGSTu with a suitable insecticide-GSH conjugate to gain further understanding of the molecular basis for detoxification of xenobiotics by this novel GST.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI; 21780049) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mishima Kaiun Memorial Foundation. This work was also supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University.
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