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Originally published In Press as doi:10.1074/jbc.M700123200 on August 22, 2007

J. Biol. Chem., Vol. 282, Issue 43, 31373-31379, October 26, 2007
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NMR Solution Structure of Lipocalin-type Prostaglandin D Synthase

EVIDENCE FOR PARTIAL OVERLAPPING OF CATALYTIC POCKET AND RETINOIC ACID-BINDING POCKET WITHIN THE CENTRAL CAVITY*Formula

Shigeru Shimamoto{ddagger}, Takuya Yoshida{ddagger}, Takashi Inui§, Keigo Gohda||, Yuji Kobayashi**, Ko Fujimori**, Toshiharu Tsurumura, Kosuke Aritake, Yoshihiro Urade, and Tadayasu Ohkubo{ddagger}1

From the {ddagger}Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan, §Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan, Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan, ||Computer-Aided Molecular Modeling Research Center Kansai, 1-3-12 Honjyo-cho, Higashinada-ku, Kobe, Hyogo 658-0012, Japan, and **Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Received for publication, January 5, 2007 , and in revised form, August 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2, an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD2-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long {alpha}-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH2 analog, revealed that PGH2 almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH2 binding mode. These results indicated that the two binding sites for PGH2 and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipocalin-type prostaglandin (PG)2 D synthase (L-PGDS, prostaglandin H2 D-isomerase, EC 5.3.99.2 [EC] ) (1–3) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2, a common precursor of various prostanoids, to produce PGD2 with 9-hydroxy and 11-keto groups, an endogenous somnogen (4) and a modulator of pain responses (5), in the presence of sulfhydryl compounds. L-PGDS is the only enzyme among members of the lipocalin gene family (6) that is composed of a group of lipid-transporter proteins, such as retinol-binding protein, beta-lactoglobulin, major urinary protein, aphorodisin (6–8), epididymal retinoic acid-binding protein (9), and tear lipocalin (10). L-PGDS has three Cys residues, Cys-65, Cys-89, and Cys-186, conserved among all species. Two of these Cys residues, Cys-89 and Cys-186, form a disulfide bridge, which is highly conserved among most, but not all lipocalins (6). This disulfide bridge can be removed without a significant loss of the enzymatic activity. On the other hand, Cys-65 is unique to L-PGDS, as it has never been found in other lipocalins. Moreover, the replacement of Cys-65 with Ser/Ala by site-directed mutagenesis led to complete loss of the catalytic activity of the recombinant rat (11), human, mouse, chicken (12), and bull and frog (13) enzymes, indicating that the Cys-65 residue is the key residue for the reaction catalyzed by L-PGDS.

L-PGDS is abundantly expressed in the central nervous system of various mammals, male genitals, human heart, and mouse adipocytes (14). L-PGDS is the same as beta-trace (15, 16), a major protein in the human cerebrospinal fluid (17), and is secreted actively into various body fluids, such as the plasma and seminal plasma. L-PGDS binds a large variety of ligands, such as retinoids (18, 19), biliverdin, bilirubin, thyroid hormones (20), gangliosides (21), and amyloid beta peptide (22), with high affinities (Kd = 20 nM to 2 µM). Thus, we thought that L-PGDS possesses dual functions as an extracellular lipophilic ligand-transporter protein as well as a PGD2-synthesizing enzyme (3). In a recent study the function of non-mammalian L-PGDS homologue without the catalytic Cys-65 residue serves as a carrier protein for lipophilic ligands but not as an enzyme (12).

The genetic and biochemical properties of L-PGDS have been extensively studied as described above, whereas the structural information on L-PGDS and its complexes with various ligands remain to be elucidated. To understand the molecular basis of the reaction mechanism and of the ligand recognition, we determined the solution structure of recombinant mouse L-PGDS mutant, which contains the catalytic Cys-65 residue, by NMR spectroscopy and clarified the ligand binding mode by molecular docking.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
NMR Spectroscopy—In this study we used the recombinant {Delta}1–24-C89A,C186A mouse L-PGDS mutant, in which the N-terminal signal sequence and the intermolecular disulfide bridge between Cys-89 and Cys-186 were removed, but the catalytically active Cys-65 residue was retained. This mutant shows the same catalytic activity and retinoid-binding activity as those of the wild type enzyme (19). Uniformly 15N- and 13C-labeled {Delta}1–24-C89A,C186A mouse L-PGDS mutant was prepared as reported previously (19) except that Escherichia coli BL21(DE3) cells were cultured in M9 minimal medium containing [15N]ammonium chloride (1 g/liter) and/or 13C glucose (2 g/liter) as the sole nitrogen and carbon sources. The NMR samples were prepared in 50 mM sodium phosphate of D2Oor 85% H2O, 15% D2O mixture at pH 6.5. The protein concentration was adjusted to ~1 mM in a 5-mm microcell NMR tube (Shigemi) for all NMR experiments. All two- and three-dimensional NMR experiments were performed at 30 °C on an INOVA600 spectrometer (Varian) equipped with shielded gradient triple resonance probes. The pulsed-field gradient techniques with a WATERGATE (23) were utilized in all H2O experiments for the solvent suppression. Transmitter frequencies for 1H, 15N, 13C{alpha}, aliphatic 13C, aromatic 13C, and carbonyl 13C were typically set to 4.75, 117.0, 60.8, 43.0, 125.0, and 176.0 ppm, respectively. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate was used as an external reference of 1H chemical shifts. 15N and 13C chemical shifts were indirectly calibrated from each gyromagnetic ratio (24). Backbone and side-chain assignments were obtained from two-dimensional 1H,15N HSQC, 13C,1H HSQC, three-dimensional HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CBCACO)NH, and HCCH-two-dimensional total correlation spectroscopy (25, 26). NOEs were collected from three-dimensional 15N-edited NOE (100-ms mixing time) and 13C-edited NOE (100-ms mixing time) spectra (25, 26). Backbone amide groups slowly exchanging with the solvent were identified from a series of two-dimensional 1H,15N HSQC spectra after a rapid buffer exchange to D2O. The retinoic acid binding site was investigated by two-dimensional 1H,15N HSQC spectra. The protein and the ligands were combined at a molar ratio of 1:1. All the NMR data were processed with NMRPipe (27) and analyzed with the NMRVIEW (Merck). A table containing the chemical shift assignments of L-PGDS has been deposited in the BioMagResBank data base under the accession number 10137.

The binding of L-PGDS to ligands was monitored by an NMR titration of 15N-labeled L-PGDS with unlabeled ligands using 1H,15N HSQC experiments. The combined 1H and 15N chemical shift changes over the range of the titration from 0 to 2 eq of ligands are plotted. Because of the instability of PGH2 in solution, the stable PGH2 analog, U-46619, was used to replace PGH2 for the interaction. The overall chemical structure of U-46619 is identical to PGH2 except for one endoperoxide oxygen that was replaced with carbon at C-9 (28).

Structure Calculation—NOE restraints were classified into four categories, strong, medium, weak, and very weak, corresponding to the distance restraints of 1.8–2.8, 1.8–3.4, 1.8–4.2, and 1.8–5.0 Å, respectively. The {Psi} and {varphi} torsion angle restraints were evaluated from the 15N, H{alpha}, 13C{alpha}, and 13Cbeta chemical shifts using the TALOS program (29). The restraints deduced from intramolecular hydrogen bonds of protein backbone, which were identified by H-D exchange experiments, were classified into two groups; between the amide proton and the carbonyl oxygen of 1.5–2.5 Å and between the amide nitrogen and the carbonyl oxygen of 2.5–3.5 Å (30). The initial solution structures were calculated using the distance geometry algorithm in the CNS programs (31). The structure optimization and energy minimization were achieved by a simulated annealing algorithm. The final 15 lowest energy structures were analyzed by using the MOLMOL (32) and PROCHECK programs (33). Structural statistics for the 15 structures are included in Table 1. Graphical representations were prepared using RASMOL and PyMOL. All of these structures have been deposited in the Protein Data Bank under accession code 2E4J.


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  		TABLE 1
Data collection and refinement statistics

 
Enzyme Assay—The L-PGDS activity was measured by incubating the enzyme at 25 °C for 1 min with [1-14C]PGH2 (final concentration of 1.25–40 µM) in 50 µl of 0.1 M Tris/HCl (pH 8.0) containing 1 mg/ml IgG and 1 mM dithiothreitol (11). [1-14C]PGH2 was prepared from [1-14C]arachidonic acid (2.20 GBq/mmol, PerkinElmer Life Sciences) as described previously (1). The all-trans-retinoic acid was dissolved in Me2SO to give a stock solution of 2 mM. The concentrations of all-trans-retinoic acid were determined spectroscopically based on its molar absorption coefficient, {epsilon}336, for all-trans-retinoic acid = 45,000 M-1 cm-1 (34). Various concentrations of all-trans-retinoic acid were added to the reaction mixture. Aggregates were removed by centrifugation, and the retinoic acid concentration was calibrated by spectrophotometry at 336 nm after the assay experiments. The U-46619 was dissolved in 0.1 M Tris/HCl (pH 8.0) to give a stock solution of 2 mM.

Molecular Modeling—Docking of retinoic acid and PGH2 to solution structure of L-PGDS was performed with AutoDock, Version 3.0 (35). This program allows ligand structures to dock in a conformationally flexible manner to a protein and adopts a rigid-body protein approximation to speed up the calculation of binding free energy. The initial structures of the ligands were constructed by the Maestro molecular modeling package (Schrödinger Inc., Portland, OR). The AMBER (36) and Gasteiger-Marsili (37, 38) atomic charges were loaded on the protein and ligand atoms as partial charges, respectively. A docking space of the ligands was defined with a rectangular box (33.8 x 45.0 x 33.8 Å3) that covers the interior space of beta-barrel of the protein. Grids of probe-atom interaction energies were computed on the boxes. The number of grid points in the x, y, and z axis was 90 x 120 x 90 with grid points separated by 0.375 Å.


Figure 1
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  		FIGURE 1.
Solution structure of L-PGDS. A, the superposition of 15 lowest-energy backbone conformers of L-PGDS. B, ribbon representation showing the typical lipocalin fold of L-PGDS, in which the secondary structural elements are labeled. C, illustration of the L-PGDS cavity. Inner accessible surface of the cavity with the polypeptide backbone is shown in gray. The ligand pocket is about 17 Å deep, 16 Å wide, and 8 Å thick and is bifurcated with 2 pockets. Pocket 1 is hydrophilic, and pocket 2 is hydrophobic. D, illustration of the cavity of representative lipocalin, retinoic acid-binding protein, for comparison. Inner accessible surface of the cavity with the polypeptide backbone is shown in gray.

 
The ligand probes were then docked by Lamarckian genetic algorithm. Flexibility was allowed for all exocyclic torsion angles of the ligands by freely rotating the torsional bonds. One Lamarckian genetic algorithm calculation generated 270,000 conformers by varying the molecular position in the docking space and torsion angles of the ligand and ranked the conformers by their binding free energy. Each docking experiment consisted of a series of 200 Lamarckian genetic algorithm simulations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structure Determination—We determined the solution structure of the {Delta}1–24-C89A,C186A mouse L-PGDS, which showed the same catalytic activity and retinoid binding activity as those of the wild type enzyme (19) by using NMR-derived distance constraints. Nearly complete assignments for backbone and side-chain 1H, 13C, and 15N resonances of L-PGDS were accomplished. The detailed restraint data for the structure calculation of L-PGDS are summarized in Table 1. A total of 1653 NOE restraints were employed for this structure calculation, including 706 intraresidue, 428 sequential, and 147 medium-range (i-i+2, i-i+3, i-i+4) and 372 long-range restraints. In addition, the 348 dihedral angle restraints were obtained from the TALOS program, and the 80 hydrogen bond restraints were observed from H-D exchange experiments. Using the CNS program, 4000 structures were calculated. The 15 structures of the lowest total energy structures with no distance restraint violations greater than 0.5 Å, and no torsion angle restraint violations above 5° were selected for further analysis as shown in Fig. 1A. Ramachandran {varphi}-{Psi} plots for the ensemble of 15 structures indicate that 72.8% of the non-glycine and non-proline residues are found in the most favored region, and 25.4% were found in the additional allowed regions. Several residues in the N terminus are found around the border of the disallowed region probably due to the lack of experimental information on the {varphi}-{Psi} angle values (39). A ribbon diagram of a representative lowest energy NMR structure of L-PGDS is shown in Fig. 1B. The superposition of backbone atoms clearly shows that the calculated structures were well converged (Fig. 1A). The overall average r.m.s.d. values to the mean structure for backbone heavy atoms and all heavy atoms including side chains of L-PGDS (residue 25–189) were 1.24 ± 0.25 and 1.73 ± 0.23 Å, respectively. The average r.m.s.d. values for backbone heavy atoms and all heavy atoms in the regular secondary structure elements were 0.41 ± 0.07 and 0.85 ± 0.07 Å, respectively.


Figure 2
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  		FIGURE 2.
The interactions of the L-PGDS with retinoic acid (A) and U-46619 (B). The composite 1H and 15N chemical shift differences versus the amino acid sequence. The composite chemical shift differences were calculated according to the empirical equation {Delta}{delta}tot = {({Delta}{delta}HN x WHN)2 + ({Delta}{delta}N xWN)2}1/2 where {Delta}{delta}HN and {Delta}{delta}N are the chemical shift changes of 1H and 15N, respectively. The weighting factors used were WHN = 1 and WN = 0.2. In both panels the residues with relatively large changes in chemical shift ({Delta}{delta}tot ≥ 0.08) are represented by a red bar. Asterisks (*) indicate the catalytic residue Cys-65. Mapping of NMR signal perturbation on L-PGDS backbone structure by binding of retinoic acid (C) and PGH2 analog, U-46619 (D). Backbone residues with relatively large changes in chemical shift ({Delta}{delta}tot ≥ 0.08) are shown in red, whereas residues with shifts in the middle range (0.06 ≤ {Delta}{delta}tot < 0.08) are shown in yellow.

 
Structure Overview of L-PGDS—L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel with a repeated +1 topology. Secondary structural elements of L-PGDS are comprised of nine beta-strands (A, amino acid residues 41–50; B, 63–71; C, 76–84; D, 89–98; E, 104–107; F, 115–121; G, 128–135; H, 144–149; I, 177–179) plus two short 310 helices (H1, 36–39; H2, 56–60) and one long {alpha}-helix (H3, 157–171) (Fig. 1B). In NMR ensemble structures of L-PGDS, the first eight residues at the N terminus, the EF loop, and GH loop are less defined due to the HN signal broadening of these loop regions. Therefore, these regions are indicated by the dashed line in Fig. 1B. The hydrophobic cavity exists in the barrel, which consists of eight strictly antiparallel beta-sheets and is largely open at one end (Fig. 1C). Four relatively flexible loops connect neighboring beta-strands in a pairwise manner at the end of the barrel. The cavity has an unusually bifurcated shape and a larger entry than those in other lipocalins. The size of the cavity is about 17 Å deep, 16 Å wide, and 8 Å thick. Analysis of the inner accessible surface of the cavity shows that there are two distinct pockets adjacent to each other, pocket 1 and pocket 2. Pocket 1 is located at the middle of the cavity, and pocket 2 is located at a relatively lower part of the cavity. For comparison, the cavity of representative lipocalins, retinoic acid-binding protein is illustrated in Fig. 1D, and the size of the cavity is about 17 Å deep, 8 Å wide, and 8 Å thick.

Recently, the crystal structure of {Delta}1–24-C65 mouse L-PGDS without enzyme activity has been deposited to the Protein Data Bank (codes 2CZT and 2CZU). Comparison of the crystal and solution structures of mouse L-PGDS revealed that both structures were similar to each other and are characterized by their overall profiles with a large cavity that consisted of two pockets as described above.

Mapping of the Binding Sites by Chemical Shift Perturbation—The ligand interaction sites of L-PGDS were determined using the NMR chemical shift perturbation method (40) in which two-dimensional 1H,15N HSQC spectra of L-PGDS were recorded with successive addition of all-trans-retinoic acid or PGH2 analog, U-46619 (Fig. 2, A and B). To allow quantification of the observed chemical shift changes, backbone resonances of L-PGDS in the complexes with retinoic acid and U-46619 were assigned (supplemental Table S1). Fig. 2A presents the magnitude of the composite 1H and 15N chemical shift differences of backbone amides observed between free and retinoic acid-bound forms of L-PGDS displayed on the primary sequence, whereas Fig. 2C presents the structural mapping of these differences. Upon retinoic acid binding, large chemical shift changes (>0.08) were observed at the region containing residues Ile-115, His-116, and Ser-117 (beta-strand F) and containing Leu-131, Phe-132, and Gly-135 (beta-strand G) (Fig. 2A). These residues form a well defined, spatially close region on the beta-barrel (Fig. 2C). A continuous, positively charged surface for binding to the carboxyl group of retinoic acid is formed on beta-strands G and H. Therefore, beta-strands F and G should be the major contact sites of the retinoic acid. On the other hand, Fig. 2B presents the composite 1H and 15N chemical shift differences of backbone amides observed between free and U-46619-bound forms of L-PGDS, whereas Fig. 2C two-dimensional presents the structural mapping of these differences. Upon U-46619 binding, large chemical shift changes (>0.08) were observed at the region containing residues His-116, Ser-117 (beta-strand F) and residues Phe-132, Ser-133, Gly-135 (beta-strand G) (Fig. 2B), similar to those observed for the retinoic acid binding. In addition, residues around Cys-65 catalytic center, Ala-60, Leu-62, Met-64, Cys-65, Thr-82, Asn-87, and Tyr-149 also show large chemical shift changes (>0.08). These residues are located around pocket 1 in the solution structure of L-PGDS (Fig. 2D). beta-Strands F and G and pocket 1 should contain the major contact sites for U-46619. Therefore, U-46619 binds in a different position from retinoic acid.


Figure 3
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  		FIGURE 3.
Docking model of L-PGDS with retinoic acid and PGH2. A, docking of retinoic acid to L-PGDS. In ribbon representation of L-PGDS backbone, the red region represents large changes observed in chemical shift ({Delta}{delta}tot ≥ 0.08) by NMR titration of L-PGDS to retinoic acid. Retinoic acid (blue) can be docked into the cavity and almost fully occupies pocket 2. The binding site estimated by docking calculations matches well with the results obtained by NMR. B, bottom of pocket 2 with docked retinoic acid. Cyclohexene ring of docked retinoic acid is located at the bottom of pocket 2 and surrounded by several aromatic residues. C, docking of PGH2 to L-PGDS. In the ribbon representation of L-PGDS backbone, the red region represents large changes observed in chemical shift ({Delta}{delta}tot ≥ 0.08) by NMR titration of L-PGDS to U-46619. PGH2 (purple) can be docked into the cavity and almost fully occupies pocket 1. D, bottom of pocket 1 with docked PGH2. The thiol group of catalytic residue Cys-65 can be in close contact with the peroxide group of PGH2.

 
Kinetic Studies of L-PGDS Mutants—To clarify the difference between U-46619 binding and PGH2 binding, the inhibition of L-PGDS reaction by U-46619 was investigated. Lineweaver-Burk plots of L-PGDS activity in the presence of various concentrations of U-46619 (see Fig. 4B) revealed an unchanged y axis intercept and an altered x axis, indicating that U-46619 inhibited L-PGDS in a competitive manner. This result clearly shows that U-46619 binds to the catalytic site of L-PGDS in the same manner as PGH2.

To identify the residues important for the PGH2 binding, the catalytic activities of several mutants, C89A,C186A/S45A, C89A,C186A/T67A, and C89A,C186A/S81A, were measured. As shown in Table 2, the mutants C89A,C186A/S45A and C89A,C186A/S81A showed a comparable Km value to C89A, C186A, whereas the mutant C89A,C186A/T67A showed a 5–6-fold higher Km value. This indicates that Thr-67 is important for the recognition of PGH2. In addition, Ser45, Thr-67, and Ser-81 are not important for retinoic acid binding because these mutants have nearly the same Kd values for retinoic acid to C89A,C186A. These results suggest that the region around Cys-65 is related to PGH2 binding but not to retinoic acid binding. The catalytic activities and Kd values for retinoic acid of mutants C89A,C186A/H116E and C89A, C186A/H116A were measured (Table 2). Both Km and Kd values of mutant C89A,C186A/H116E were higher than that of C89A,C186A. The mutant C89A,C186A/H116A showed a 3-fold higher Km value but almost the same Kd value for retinoic acid. These results may indicate that the positive charge around His-116 contributes to the PGH2 and retinoic acid binding and that His-116 is involved in PGH2 binding but not in retinoic acid binding.


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  		TABLE 2
Kinetic parameters of mouse L-PGDS mutants

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Unusually Bifurcated Cavity of L-PGDS—Fig. 1C clearly shows that the cavity of L-PGDS has an unusually bifurcated shape and a larger entry than those in other lipocalins. Analysis of the inner accessible surface shows that there are two distinct pockets adjacent to each other. Pocket 1 is located at the middle of the cavity, and pocket 2 is located at a relatively lower part of the cavity. Most of the residues located around the pocket 2 are aromatic and hydrophobic. In contrast, the pocket 1 is surrounded by several polar residues such as Ser or Thr and is more hydrophilic than the pocket 2. The two pockets are separated by residues Tyr-149 and Thr-67. These data indicate that the two binding pockets in the large cavity of L-PGDS should be responsible for the broad ligand specificity of L-PGDS.


Figure 4
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  		FIGURE 4.
A, overlay of the retinoic acid-docking model and the PGH2-docking model. Kinetic analysis of L-PGDS inhibition by the PGH2 analog, U-46619 (B), and retinoic acid (C) is shown. Lineweaver-Burk plots of L-PGDS activity in the presence of various concentrations of U-46619 and retinoic acid are shown. L-PGDS was incubated with 1.25–40 µM PGH2 and 1 mM dithiothreitol. Data points were fitted using least square analysis.

 
Molecular Docking Studies—To explore the ligand binding mode, we constructed two molecular models bound to retinoic acid or PGH2. In the retinoic acid-docking model (Fig. 3A), the retinoic acid lies on the beta-strand F and G, and the cyclohexene ring of retinoic acid can be placed into the hydrophobic pocket 2. Structural comparison with other retinoid binding lipocalins showed that aromatic residues important for retinoid binding are also conserved in L-PGDS. These aromatic residues, Phe-34, Phe-39, Tyr-105, and Tyr-149, are well defined (the side chain r.m.s.d. values = 0.4–0.7 Å) for L-PGDS and make up the aromatic cluster around the pocket 2 at the bottom of the beta-barrel. The distances between these aromatic residues of L-PGDS and the beta-ionone ring are within 4 or 5 Å, indicating the relatively strong van der Waals contacts (Fig. 3B). Corresponding distances in the crystal structure of retinoic acid-binding protein-retinoic acid complex (Protein Data Bank code 1EPA [PDB] ) also rang from 3.0 to 4.4 Å. Therefore, the aromatic cluster of L-PGDS, located at the bottom of the pocket 2, is considered to be important for retinoid binding. Both L-PGDS (Kd = 80–150 nM) and retinoic acid-binding protein (Kd = 900 nM) exhibit relatively strong affinity to retinoic acid. In the docking model, an aliphatic side chain of retinoic acid is pointed toward the open entry of the beta-barrel. The terminal carboxylate group is located in the vicinity of the positively charged region (beta-strand F). This retinoic acid binding model is consistent with the results of NMR titration experiments and mutant analysis.

In the PGH2-docking model (Fig. 3C), the PGH2 was in the middle of the cavity, and the cyclopentane ring of PGH2 almost completely occupied pocket 1. In this model the thiol group of Cys-65 is located within a 5-Å distance from the oxygen atoms of the 9,11-endoperoxide group of PGH2 (Fig. 3D), indicating that these atoms are in contact distance with each other. This is consistent with the previously proposed PGH2 reaction mechanism by Urade et al. (11). In the proposed mechanism, conversion of PGH2 is initiated by a nucleophilic attack of the active intrinsic thiol of Cys-65 to the oxygen at C-11 of PGH2. The extrinsic sulfhydryl group withdraws the hydrogen at C-11 to produce PGD2 in the following reaction step. Ser-45, Thr-67, and Ser-81 residues were close to oxygen atoms of the endoperoxide group of PGH2 with distances of about 4 Å (Fig. 3B). Biochemical studies of the cane toad homolog of L-PGDS suggested that these residues may be involved in the recognition of the oxygen atoms of the endoperoxide group of PGH2 (13). Present mutant analyses indicated that Thr-67 is the key residue among these residues. The cyclopentane ring and {alpha} and {omega} chains of PGH2 were not fixed in their spatial relationship in solution. However, the {alpha} chain, with a negative charge at the carboxyl group, turned on the positive-charged region arising from His-116 and Lys-137 located at the edge of the cavity of L-PGDS. This PGH2 binding model supports that PGH2 binds to the region around the Cys-65 in the catalytic pocket 1, which is suggested by the U-46619 binding experiments. Interestingly, the overall structure of PGH2 in our complex model is similar to the previously reported free form structure of U-46619 in a membrane-mimicking environment (28).

Non-competitive Inhibition of Retinoic Acid—An overlay of the retinoic acid-docking model and the PGH2-docking model is shown in Fig. 4A. The terminal portions of the aliphatic chain of retinoic acid and the {alpha} chain of PGH2 are slightly overlapped (yellow circle). In both binding models of retinoic acid and PGH2, there is a possibility that each of the carboxyl groups competes for His-116. Mutational studies of His-116 revealed that the mutant H116A had a nearly comparable Kd value for retinoic acid but showed about a 2–3-fold higher Km value. These results indicate that His-116 was important for the recognition of PGH2 but not for retinoic acid. This observation prompted us to investigate the inhibition kinetic of L-PGDS by retinoic acid. Lineweaver-Burk plots of L-PGDS activity in the presence of various concentrations of all-trans-retinoic acid (Fig. 4C) revealed the unchanged an x axis intercept and an altered y axis, indicating that all-trans-retinoic acid inhibited L-PGDS in a non-competitive manner. Although retinoic acid was bound to L-PGDS with the strong affinity of Kd of 80–150 nM (18, 19), all-trans-retinoic acid weakly inhibited the L-PGDS activity with the estimated Ki value of 5–6 µM in a non-competitive manner against PGH2; this is consistent with the current docking model (Fig. 3C). These results provide the direct evidence for two binding sites for PGH2 and retinoic acid in the large cavity of L-PGDS.

The solution structure described here is in good agreement with the previously reported structural information from the fluorescence quenching assay and mutational studies (19). These data revealed that the Trp-43 residue contributed to the fluorescence quenching of L-PGDS upon lipophilic ligand binding. The structure of L-PGDS shows that Trp-43 is located at the bottom of the hydrophobic cavity near pocket 2 and can interact with ligands. L-PGDS can bind various structurally different hydrophobic ligands, such as retinoids (18, 19), biliverdin, bilirubin, thyroid hormones (18, 20), gangliosides (21), and amyloid beta peptide (22). The larger cavity of L-PGDS makes it possible to bind bulky compounds such as bilirubin or biliverdin. The unusually shaped and large cavity of L-PGDS, therefore, reflects the broad selectivity of ligand binding.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2E4J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The chemical shift assignments for free form of L-PGDS have been deposited in the BioMagRes Data Bank (www.bmrb.wisc.edu) under the accession number BMRB 10137.

* This work was supported in part by of Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research 18054021 (to T. O.) and 19590094 (to K. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed. Tel.: 81-6-6879-8223; Fax: 81-6-6879-8221; E-mail: ohkubo{at}phs.osaka-u.ac.jp.

2 The abbreviations used are: PG, prostaglandin; L-PGDS, lipocalin-type PG synthase; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; r.m.s.d., root mean square deviation. Back


   ACKNOWLEDGMENTS
 
We thank M. Sakata of Osaka Bioscience Institute for assistance in enzyme assay and binding analysis and M. Inui, M. Fujii, and K. Nakajima of Osaka University for technical assistance in the early stage of NMR study.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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