- Email: [email protected]
Crystal structure of lipocalin-type prostaglandin D synthase
International Congress Series 1233 (2002) 453 – 459
Crystal structure of lipocalin-type prostaglandin D synthase Daisuke Irikura a,b, Takashi Kumasaka c, Masaki Yamamoto c, Osamu Hayaishi b, Yoshihiro Urade a,b,* a
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, c/o Osaka Bioscience Institute, Osaka 565-0874, Japan b Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan c RIKEN Harima Institute, Mikazuki, Sayo, Hyogo 679-5148, Japan
Abstract Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, a potent endogenous sleep-inducing substance found in the central nervous system. In this study, we crystallized recombinant mouse L-PGDS from a sodium citrate solution by the hanging drop vapor-diffusion method. The crystal structure of the selenomethionyl Cys65Ala-substituted L˚ resolution by using the multiwavelength anomalous diffraction PGDS was determined at 2.5 A method. The crystals belonged to the orthorhombic space group C2221 with lattice constants ˚ . The overall architecture of L-PGDS had an eight-stranded h-barrel a = 45.5, b = 66.8, c = 104.5 A with a hydrophobic cavity, in which the Cys65 residue, an active site for the catalysis, was identified. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lipocalin; Prostaglandin D synthase; Prostaglandin D2; Crystal structure; Multiwavelength anomalous diffraction method
1. Introduction Prostaglandins (PGs) are potent biological mediators derived from arachidonic acid and are formed in response to a variety of immunologic and inflammatory stimuli. PGD2 is the Abbreviations: L-PGDS, lipocalin-type prostaglandin D synthase; H-PGDS, hematopoietic prostaglandin D synthase; PGD2, prostaglandin D2; PGH2, prostaglandin H2; MAD, multiwavelength anomalous diffraction; DLS, dynamic light scattering. * Corresponding author. Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: +81-6-6872-4851; fax: +81-6-6872-2841. E-mail address: [email protected] (Y. Urade). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 2 7 - 7
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major PG produced in the central nervous system of various mammals and is well known as an endogenous sleep-promoting substance [1]. PGD2 also regulates nociceptive responses [2] and is responsible for the symptoms in patients with mastocytosis [3] and African sleeping sickness [4]. PGD synthase (PGDS, EC 5.3.99.2) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2, a common precursor of various prostanoids, to produce PGD2 having 9-hydroxy and 11-keto groups in the presence of sulfhydryl compounds. There are two evolutionally distinct types of PGDS [5]; one is the lipocalin-type enzyme (L-PGDS) [6] and the other is the hematopoietic one (H-PGDS) [7]. H-PGDS contributes to production of PGD2 in the peripheral tissues. Molecular evolutional studies have revealed that this enzyme is the vertebrate homolog of the sigma class glutathione S-transferase. We previously reported that the recombinant rat H-PGDS crystallized from a polyethylene ˚ resolution by the glycol 6000 solution, and determined the crystal structure at 2.3 A multiple isomorphous replacement method. The overall structure of H-PGDS was found to be an a-helix-rich structure consisting of two domains with a prominent interdomain cleft. The cleft including the glutathione-binding site was assumed to be the catalytic pocket [7]. On the other hand, L-PGDS is responsible for the production of PGD2 in the central nervous system of various mammals. Amino acid sequence analysis of L-PGDS revealed that this enzyme is a member of the lipocalin superfamily composed of various secretory lipophilic-transporter proteins. When selenium chloride, a relatively selective and reversible inhibitor of L-PGDS, was infused into the brain of rats during the daytime, sleep was almost completely and reversibly inhibited in a time- and dose-dependent manner [8]. Moreover, we recently reported that transgenic mice overexpressing human L-PGDS exhibited excessive amounts of non-rapid eye movement sleep in response to the noxious stimulus by tail clipping, coupled with a significant increase in PGD2 production in the brain [9]. Therefore, PGD2 and L-PGDS are considered to play an important role in the regulation of sleep, especially non-rapid eye movement sleep. However, the structure and mechanism of the enzymatic reaction of L-PGDS remain to be elucidated. In this study, we crystallized recombinant L-PGDS and determined its crystal structure by the multiwavelength anomalous diffraction (MAD) method.
2. Materials and methods 2.1. Expression and purification of recombinant L-PGDS To efficiently obtain the correctly folded recombinant mouse L-PGDS, we used the Cys65Ala-substituted L-PGDS without the 22-amino acid N-terminal signal peptide [10]. The cDNA for mouse L-PGDS was cloned into the expression vector pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden), which was then used to transfect to Escherichia coli DH5a (TOYOBO, Tokyo, Japan). The recombinant L-PGDS was expressed as a glutathione S-transferase-fusion protein and purified by glutathione – Sepharose 4B column chromatography (Amersham Pharmacia Biotech) after incubation with thrombin (Sigma-Aldrich, WI, USA). L-PGDS was further purified by column
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chromatography with Superdex75 (Amersham Pharmacia Biotech) and then Mono-S (Amersham Pharmacia Biotech), as reported previously [10,11]. Selenomethionyl L-PGDS used for the MAD method was obtained from E. coli B834 (DE3) (Novagen, WI, USA) transformed with the mouse L-PGDS cDNA-containing pGEX2T vector, which bacteria were cultured in the chemically defined, selenomethioninecontaining amino acid-enriched medium. The purified enzyme was dialyzed against 5 mM Tris/HCl (pH 8) containing 10 AM all-trans retinoic acid (Sigma-Aldrich), and concentrated up to 10 mg/ml by ultrafiltration with a YM-3 membrane (Millipore, MA, USA). 2.2. Crystallization and collection of X-ray diffraction data Size distribution of the protein molecules in the solution was assessed by dynamic light-scattering (DLS) spectroscopy with a DynaPro DLS instrument (PROTEIN SOLUTIONS, VA, USA). Crystallization experiments were carried out at a constant temperature of 22.5 jC by the hanging drop vapor-diffusion method using 24-well tissue culture trays (ICN, OH, USA) as described in Section 3. To optimize the anomalous effect, we measured the X-ray fluorescence spectrum with a selenomethionyl crystal near the absorption edge of selenium. For the MAD method [12], selenomethionyl crystals were harvested directly from the crystallization drop, soaked in a cryoprotectant solution prepared by addition of a final concentration of 0.5 M trehalose to the crystallization solution, and flash frozen at 100 K. The MAD data were collected by using the inversebeam method on SPring-8 (BL45XU-PX) [13], and processed and scaled with software of DENZO and SCALEPACK, respectively [14]. Phase refinement and density modification were performed with software of SHARP [15] and SOLOMON, respectively [16]. The electron density map was displayed by the Program O [17] to build the model of L-PGDS. Crystallographic refinement was performed by using alternating cycles of CNS [18] refinement.
3. Results Among the various lipophilic ligands for L-PGDS examined, all-trans retinoic acid was found to be effective to maintain a monodispersed state of L-PGDS molecules in the sample solution. The DLS experiments demonstrated that the L-PGDS preparation in the presence of an excess amount of all-trans retinoic acid (10 AM) was monodisperse, with a monomodal distribution of size Rh = 2.1 nm and a polydispersity of less than 0.1 nm. These results also indicated that the solution did not contain measurable impurities or aggregates. The purified L-PGDS protein was crystallized even after storage at 4 jC for 4 years in the presence of all-trans retinoic acid. The Cys65Ala-substituted L-PGDS was crystallized by mixing the protein solution (2 Al) with an equal volume of reservoir solution containing 1.25 M sodium citrate, 0.1 M Tris/HCl (pH 9.5), and 10% (v/v) 1,4-dioxane. The mixture (4 Al) was equilibrated against the reservoir solution (1 ml). The selenomethionyl Cys65Ala L-PGDS was also crystallized under the same conditions.
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Fig. 1. X-ray fluorescence spectrum of a selenomethionyl crystal of L-PGDS. This figure shows the near absorption edge of a selenomethionyl crystal of L-PGDS.
Fig. 2. Crystal structure of mouse L-PGDS. Ribbon diagram of L-PGDS produced by MOLSCRIPT [20]. Cys65 as an active site for the enzymatic reaction, drawn in the ball and stick style.
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We obtained native Cys65Ala crystals with a maximum dimension of 0.1 0.1 0.4 mm3 within 3 weeks. All crystals belonged to the orthorhombic space group C2221 with ˚ and containing one molecule per the cell dimensions a = 45.5, b = 66.8, c = 104.5 A asymmetric unit. X-ray fluorescence spectra were measured at SPring-8 BL45XU-PX. Fig. 1 shows the spectrum of the selenomethionyl crystals of L-PGDS near the absorption edge. Peak and ˚ , respectively. We collected diffraction data edge were observed at 0.9797 and 0.9803 A ˚ ). By using the with three different wavelengths of the peak, edge and remote (1.100 A diffraction data, we detected three selenium sites in the Patterson maps. These selenium sites, which were assigned by using anomalous difference Fourier maps, were helpful in building the model. The model of L-PGDS structure consists of a single domain made up by an eightstranded antiparallel h-barrel with a hydrophobic cavity. The Cys65 residue of L-PGDS, an active site for catalysis, was located on the inside wall of the hydrophobic cavity (Fig. 2).
4. Discussion In this study, we determined the crystal structure of the selenomethionyl Cys65Ala mutant of mouse L-PGDS by the MAD method. The PGDS activity of the purified selenomethionyl Cys89,186Ala mutant (5 Amol/min/mg of protein) was almost the same as that activity of the native Cys89,186Ala enzyme containing methionine instead of selenomethionine. Furthermore, the elution profiles of Cys89,186Ala and Cys65Ala mutants from Superdex75 and Mono-S column were essentially identical to the native and selenomethionyl proteins. These results suggest that the overall architecture of selenomethionyl L-PGDS is the same as that of the native enzyme. An initial screening of crystallization conditions with the purified recombinant mouse Cys65Ala-substituted L-PGDS in the absence of lipophilic ligands was not successful. DLS spectroscopy revealed that the purified enzyme did not monodisperse in the absence of lipophilic ligands. All-trans retinoic acid was found to be effective to maintain a monodisperse state of L-PGDS molecules, as monitored by the DLS analysis. Bilirubin and biliverdin are also bound by L-PGDS at lower Kd value (30 – 40 nM) than those of retinoids (70 – 80 nM) [11]. However, bilirubin and biliverdin were not effective to maintain a monodisperse state and, thus, the crystallization of L-PGDS in the presence of these ligands was not successful. Although the L-PGDS was crystallized in the presence of all-trans retinoic acid, the molecule was not observed in the electron density, suggesting that the location of retinoic acid molecule was flexible in the hydrophobic cavity. Compared with other lipocalins whose tertiary structures have already been determined [19], such as retinol-binding protein, epididymal retinoic acid-binding protein, major urinary protein, odorant-binding protein, h-lactoglobulin, bilin-binding protein and Nitrophorin, the overall structure of L-PGDS was almost the same as that of the other lipocalins. Especially, the depth and width of the hydrophobic cavity of L-PGDS were similar to those of major urinary protein, suggesting that the h-barrel structure has been maintained during the evolution from the common ancestral lipocalin protein to L-PGDS and major urinary protein.
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However, the crystal structure of L-PGDS (h-barrel structure) was quite different from that of H-PGDS [7], we had previously determined to be an a-helix-rich one, although both enzymes catalyze the same reaction from PGH2 to PGD2. The crystal structures of LPGDS and H-PGDS are useful to design selective and/or nonselective inhibitors for each enzyme that may act as antisomnolence and antiinflammatory drugs.
Acknowledgements We thank Prof. T. Tsukihara (Osaka University), Dr. M. Miyano (RIKEN Harima) and Dr. H. Ago (JAPAN TABACCO) for guidance of the protein crystallization method. We also thank Prof. S. Uemura (Kyoto University) for the chemical synthesis of selenomethionine. This study was supported in part by grants from the CREST Project, Japan Science and Technology; the Ground Research for Space Utilization promoted by NASDA and the Japan Space Forum; the Japan Foundation for Applied Enzymology; and Osaka City.
References [1] Y. Urade, O. Hayaishi, Prostaglandin D2 and sleep regulation, Biochim. Biophys. Acta 1436 (1999) 606 – 615. [2] N. Eguchi, T. Minami, N. Shirafuji, Y. Kanaoka, T. Tanaka, A. Nagata, N. Yoshida, Y. Urade, S. Ito, O. Hayaishi, Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 726 – 730. [3] L.J. Roberts 2nd, B.J. Sweetman, R.A. Lewis, K.F. Austen, J.A. Oates, Increased production of prostaglandin D2 in patients with systemic mastocytosis, N. Engl. J. Med. 303 (1980) 1400 – 1404. [4] V.W. Pentreath, K. Rees, O.A. Owolabi, K.A. Philip, F. Doua, The somnogenic T lymphocyte suppressor prostaglandin D2 is selectively elevated in cerebrospinal fluid of advanced sleeping sickness patients, Trans. R. Soc. Trop. Med. Hyg. 84 (1990) 795 – 799. [5] Y. Urade, O. Hayaishi, Prostaglandin D synthase: structure and function, Vitam. Horm. 58 (2000) 89 – 120. [6] Y. Urade, O. Hayaishi, Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase, Biochim. Biophys. Acta 1482 (2000) 259 – 271. [7] Y. Kanaoka, H. Ago, E. Inagaki, T. Nanayama, M. Miyano, R. Kikuno, Y. Fujii, N. Eguchi, H. Toh, Y. Urade, O. Hayaishi, Cloning and crystal structure of hematopoietic prostaglandin D synthase, Cell 90 (1997) 1085 – 1095. [8] H. Matsumura, R. Takahata, O. Hayaishi, Inhibition of sleep in rats by inorganic selenium compounds, inhibitors of prostaglandin D synthase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 9046 – 9050. [9] E. Pinzar, Y. Kanaoka, T. Inui, N. Eguchi, Y. Urade, O. Hayaishi, Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4903 – 4907. [10] Y. Urade, T. Tanaka, N. Eguchi, M. Kikuchi, H. Kimura, H. Toh, O. Hayaishi, Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. Identification of Cys65 as an essential thiol, J. Biol. Chem. 270 (1995) 1422 – 1428. [11] C.T. Beuckmann, M. Aoyagi, I. Okazaki, T. Hiroike, H. Toh, O. Hayaishi, Y. Urade, Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase, Biochemistry 38 (1999) 8006 – 8013. [12] W.A. Hendrickson, J.R. Horton, D.M. LeMaster, Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure, EMBO J. 9 (1990) 1665 – 1672. [13] M. Yamamoto, T. Kumasaka, T. Fujisawa, T. Ueki, Trichromatic concept at SPring-8 RIKEN beamline I, J. Synchrotron Rad. 5 (1998) 222 – 226.
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[14] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation mode, Methods Enzymol. 276 (1997) 307 – 326. [15] E. de La Fortelle, G. Bricogne, Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods, Methods Enzymol. 276 (1997) 472 – 494. [16] J.P. Abrahams, A.G.W. Leslie, Methods used in the structure determination of bovine mitochondrial F1 ATPase, Acta Crystallogr., Sect. D. 32 (1996) 30 – 42. [17] T.A. Jones, J.-Y. Zou, S. Cowan, Improved methods for building protein models in electron density maps and the location for errors in these models, Acta Crystallogr., Sect. A. 47 (1991) 110 – 119. [18] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. De Lano, P. Gros, R.W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography and NMR System: a new software suite for macromolecular structure determination, Acta Crystallogr., Sect. D 54 (1996) 905 – 921. [19] D.R. Flower, Experimentally determined lipocalin structures, Biochim. Biophys. Acta 1482 (2000) 46 – 56. [20] P.J. Kraulis, MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure, J. Appl. Crystallogr. 24 (1991) 946 – 950.
Crystal structure of lipocalin-type prostaglandin D synthase Daisuke Irikura a,b, Takashi Kumasaka c, Masaki Yamamoto c, Osamu Hayaishi b, Yoshihiro Urade a,b,* a
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, c/o Osaka Bioscience Institute, Osaka 565-0874, Japan b Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan c RIKEN Harima Institute, Mikazuki, Sayo, Hyogo 679-5148, Japan
Abstract Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, a potent endogenous sleep-inducing substance found in the central nervous system. In this study, we crystallized recombinant mouse L-PGDS from a sodium citrate solution by the hanging drop vapor-diffusion method. The crystal structure of the selenomethionyl Cys65Ala-substituted L˚ resolution by using the multiwavelength anomalous diffraction PGDS was determined at 2.5 A method. The crystals belonged to the orthorhombic space group C2221 with lattice constants ˚ . The overall architecture of L-PGDS had an eight-stranded h-barrel a = 45.5, b = 66.8, c = 104.5 A with a hydrophobic cavity, in which the Cys65 residue, an active site for the catalysis, was identified. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lipocalin; Prostaglandin D synthase; Prostaglandin D2; Crystal structure; Multiwavelength anomalous diffraction method
1. Introduction Prostaglandins (PGs) are potent biological mediators derived from arachidonic acid and are formed in response to a variety of immunologic and inflammatory stimuli. PGD2 is the Abbreviations: L-PGDS, lipocalin-type prostaglandin D synthase; H-PGDS, hematopoietic prostaglandin D synthase; PGD2, prostaglandin D2; PGH2, prostaglandin H2; MAD, multiwavelength anomalous diffraction; DLS, dynamic light scattering. * Corresponding author. Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: +81-6-6872-4851; fax: +81-6-6872-2841. E-mail address: [email protected] (Y. Urade). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 2 7 - 7
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major PG produced in the central nervous system of various mammals and is well known as an endogenous sleep-promoting substance [1]. PGD2 also regulates nociceptive responses [2] and is responsible for the symptoms in patients with mastocytosis [3] and African sleeping sickness [4]. PGD synthase (PGDS, EC 5.3.99.2) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2, a common precursor of various prostanoids, to produce PGD2 having 9-hydroxy and 11-keto groups in the presence of sulfhydryl compounds. There are two evolutionally distinct types of PGDS [5]; one is the lipocalin-type enzyme (L-PGDS) [6] and the other is the hematopoietic one (H-PGDS) [7]. H-PGDS contributes to production of PGD2 in the peripheral tissues. Molecular evolutional studies have revealed that this enzyme is the vertebrate homolog of the sigma class glutathione S-transferase. We previously reported that the recombinant rat H-PGDS crystallized from a polyethylene ˚ resolution by the glycol 6000 solution, and determined the crystal structure at 2.3 A multiple isomorphous replacement method. The overall structure of H-PGDS was found to be an a-helix-rich structure consisting of two domains with a prominent interdomain cleft. The cleft including the glutathione-binding site was assumed to be the catalytic pocket [7]. On the other hand, L-PGDS is responsible for the production of PGD2 in the central nervous system of various mammals. Amino acid sequence analysis of L-PGDS revealed that this enzyme is a member of the lipocalin superfamily composed of various secretory lipophilic-transporter proteins. When selenium chloride, a relatively selective and reversible inhibitor of L-PGDS, was infused into the brain of rats during the daytime, sleep was almost completely and reversibly inhibited in a time- and dose-dependent manner [8]. Moreover, we recently reported that transgenic mice overexpressing human L-PGDS exhibited excessive amounts of non-rapid eye movement sleep in response to the noxious stimulus by tail clipping, coupled with a significant increase in PGD2 production in the brain [9]. Therefore, PGD2 and L-PGDS are considered to play an important role in the regulation of sleep, especially non-rapid eye movement sleep. However, the structure and mechanism of the enzymatic reaction of L-PGDS remain to be elucidated. In this study, we crystallized recombinant L-PGDS and determined its crystal structure by the multiwavelength anomalous diffraction (MAD) method.
2. Materials and methods 2.1. Expression and purification of recombinant L-PGDS To efficiently obtain the correctly folded recombinant mouse L-PGDS, we used the Cys65Ala-substituted L-PGDS without the 22-amino acid N-terminal signal peptide [10]. The cDNA for mouse L-PGDS was cloned into the expression vector pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden), which was then used to transfect to Escherichia coli DH5a (TOYOBO, Tokyo, Japan). The recombinant L-PGDS was expressed as a glutathione S-transferase-fusion protein and purified by glutathione – Sepharose 4B column chromatography (Amersham Pharmacia Biotech) after incubation with thrombin (Sigma-Aldrich, WI, USA). L-PGDS was further purified by column
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chromatography with Superdex75 (Amersham Pharmacia Biotech) and then Mono-S (Amersham Pharmacia Biotech), as reported previously [10,11]. Selenomethionyl L-PGDS used for the MAD method was obtained from E. coli B834 (DE3) (Novagen, WI, USA) transformed with the mouse L-PGDS cDNA-containing pGEX2T vector, which bacteria were cultured in the chemically defined, selenomethioninecontaining amino acid-enriched medium. The purified enzyme was dialyzed against 5 mM Tris/HCl (pH 8) containing 10 AM all-trans retinoic acid (Sigma-Aldrich), and concentrated up to 10 mg/ml by ultrafiltration with a YM-3 membrane (Millipore, MA, USA). 2.2. Crystallization and collection of X-ray diffraction data Size distribution of the protein molecules in the solution was assessed by dynamic light-scattering (DLS) spectroscopy with a DynaPro DLS instrument (PROTEIN SOLUTIONS, VA, USA). Crystallization experiments were carried out at a constant temperature of 22.5 jC by the hanging drop vapor-diffusion method using 24-well tissue culture trays (ICN, OH, USA) as described in Section 3. To optimize the anomalous effect, we measured the X-ray fluorescence spectrum with a selenomethionyl crystal near the absorption edge of selenium. For the MAD method [12], selenomethionyl crystals were harvested directly from the crystallization drop, soaked in a cryoprotectant solution prepared by addition of a final concentration of 0.5 M trehalose to the crystallization solution, and flash frozen at 100 K. The MAD data were collected by using the inversebeam method on SPring-8 (BL45XU-PX) [13], and processed and scaled with software of DENZO and SCALEPACK, respectively [14]. Phase refinement and density modification were performed with software of SHARP [15] and SOLOMON, respectively [16]. The electron density map was displayed by the Program O [17] to build the model of L-PGDS. Crystallographic refinement was performed by using alternating cycles of CNS [18] refinement.
3. Results Among the various lipophilic ligands for L-PGDS examined, all-trans retinoic acid was found to be effective to maintain a monodispersed state of L-PGDS molecules in the sample solution. The DLS experiments demonstrated that the L-PGDS preparation in the presence of an excess amount of all-trans retinoic acid (10 AM) was monodisperse, with a monomodal distribution of size Rh = 2.1 nm and a polydispersity of less than 0.1 nm. These results also indicated that the solution did not contain measurable impurities or aggregates. The purified L-PGDS protein was crystallized even after storage at 4 jC for 4 years in the presence of all-trans retinoic acid. The Cys65Ala-substituted L-PGDS was crystallized by mixing the protein solution (2 Al) with an equal volume of reservoir solution containing 1.25 M sodium citrate, 0.1 M Tris/HCl (pH 9.5), and 10% (v/v) 1,4-dioxane. The mixture (4 Al) was equilibrated against the reservoir solution (1 ml). The selenomethionyl Cys65Ala L-PGDS was also crystallized under the same conditions.
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Fig. 1. X-ray fluorescence spectrum of a selenomethionyl crystal of L-PGDS. This figure shows the near absorption edge of a selenomethionyl crystal of L-PGDS.
Fig. 2. Crystal structure of mouse L-PGDS. Ribbon diagram of L-PGDS produced by MOLSCRIPT [20]. Cys65 as an active site for the enzymatic reaction, drawn in the ball and stick style.
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We obtained native Cys65Ala crystals with a maximum dimension of 0.1 0.1 0.4 mm3 within 3 weeks. All crystals belonged to the orthorhombic space group C2221 with ˚ and containing one molecule per the cell dimensions a = 45.5, b = 66.8, c = 104.5 A asymmetric unit. X-ray fluorescence spectra were measured at SPring-8 BL45XU-PX. Fig. 1 shows the spectrum of the selenomethionyl crystals of L-PGDS near the absorption edge. Peak and ˚ , respectively. We collected diffraction data edge were observed at 0.9797 and 0.9803 A ˚ ). By using the with three different wavelengths of the peak, edge and remote (1.100 A diffraction data, we detected three selenium sites in the Patterson maps. These selenium sites, which were assigned by using anomalous difference Fourier maps, were helpful in building the model. The model of L-PGDS structure consists of a single domain made up by an eightstranded antiparallel h-barrel with a hydrophobic cavity. The Cys65 residue of L-PGDS, an active site for catalysis, was located on the inside wall of the hydrophobic cavity (Fig. 2).
4. Discussion In this study, we determined the crystal structure of the selenomethionyl Cys65Ala mutant of mouse L-PGDS by the MAD method. The PGDS activity of the purified selenomethionyl Cys89,186Ala mutant (5 Amol/min/mg of protein) was almost the same as that activity of the native Cys89,186Ala enzyme containing methionine instead of selenomethionine. Furthermore, the elution profiles of Cys89,186Ala and Cys65Ala mutants from Superdex75 and Mono-S column were essentially identical to the native and selenomethionyl proteins. These results suggest that the overall architecture of selenomethionyl L-PGDS is the same as that of the native enzyme. An initial screening of crystallization conditions with the purified recombinant mouse Cys65Ala-substituted L-PGDS in the absence of lipophilic ligands was not successful. DLS spectroscopy revealed that the purified enzyme did not monodisperse in the absence of lipophilic ligands. All-trans retinoic acid was found to be effective to maintain a monodisperse state of L-PGDS molecules, as monitored by the DLS analysis. Bilirubin and biliverdin are also bound by L-PGDS at lower Kd value (30 – 40 nM) than those of retinoids (70 – 80 nM) [11]. However, bilirubin and biliverdin were not effective to maintain a monodisperse state and, thus, the crystallization of L-PGDS in the presence of these ligands was not successful. Although the L-PGDS was crystallized in the presence of all-trans retinoic acid, the molecule was not observed in the electron density, suggesting that the location of retinoic acid molecule was flexible in the hydrophobic cavity. Compared with other lipocalins whose tertiary structures have already been determined [19], such as retinol-binding protein, epididymal retinoic acid-binding protein, major urinary protein, odorant-binding protein, h-lactoglobulin, bilin-binding protein and Nitrophorin, the overall structure of L-PGDS was almost the same as that of the other lipocalins. Especially, the depth and width of the hydrophobic cavity of L-PGDS were similar to those of major urinary protein, suggesting that the h-barrel structure has been maintained during the evolution from the common ancestral lipocalin protein to L-PGDS and major urinary protein.
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However, the crystal structure of L-PGDS (h-barrel structure) was quite different from that of H-PGDS [7], we had previously determined to be an a-helix-rich one, although both enzymes catalyze the same reaction from PGH2 to PGD2. The crystal structures of LPGDS and H-PGDS are useful to design selective and/or nonselective inhibitors for each enzyme that may act as antisomnolence and antiinflammatory drugs.
Acknowledgements We thank Prof. T. Tsukihara (Osaka University), Dr. M. Miyano (RIKEN Harima) and Dr. H. Ago (JAPAN TABACCO) for guidance of the protein crystallization method. We also thank Prof. S. Uemura (Kyoto University) for the chemical synthesis of selenomethionine. This study was supported in part by grants from the CREST Project, Japan Science and Technology; the Ground Research for Space Utilization promoted by NASDA and the Japan Space Forum; the Japan Foundation for Applied Enzymology; and Osaka City.
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