Biochemical and structural characteristics of hematopoietic prostaglandin D synthase: From evolutionary analysis to drug designing

Hematopoietic prostaglandin (PG) D sythase (HPGDS) catalyzes the isomerization of PGH2, a common intermediate of various prostanoids, to PGD2, an inflammatory mediator, in the presence of glutathione (GSH). H-PGDS is activated by Mg, which increases the affinity of the enzyme for GSH and the turnover number. An evolutionary study revealed H-PGDS to be Correspondence/Reprint request: Dr. Yoshihiro Urade, Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan. E-mail: [email protected] Yoshihiro Urade et al. 136 the first identified mammalian member of the sigma class of GSH-transferases. The crystallographic analysis of the rat and human enzymes identified a prominent cleft near the bound GSH to be the catalytic center. In the human enzyme, the Mg ion is octahedrally coordinated by 6 water molecules at the interface of a homodimer, in which Asp93, Asp96, and Asp97 from each subunit surround 6 water molecules. An orally effective H-PGDS inhibitor, 4benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]-piperidine (HQL-79) was found to bind within the catalytic cleft between Trp104 and GSH. Oral administration of HQL-79 suppressed antigen-induced eosinophilic accumulation in the lung of wild-type mice and human H-PGDSoverexpressing mice, gliosis and demyelination in twitcher mice, and muscular distrophy in mdx mice. The tertiary structure of H-PGDS gives informative clues for the development of inhibitors specific for this enzyme, of which inhibitors are promising drugs to suppress allergic inflammation, neuroinflammation, and muscular dystrophy. Introduction Prostaglandin (PG) D2 is a mediator of allergic and inflammatory responses produced by mast cells (1) and Th2 cells (2) in a variety of tissues, and is also an endogenous somnogen acting within the brain (3). PGD2 is formed from arachidonic acid by successive enzyme reactions (Fig. 1): PG endoperoxide synthase (cyclooxygenase, COX) catalyzes the di-oxygenation of arachidonic acid to PGH2 via PGG2, and PGD synthase (PGDS) then catalyzes the isomerization of PGH2 to PGD2 (3). There are 2 distinct types of PGDS: one is hematopoietic PGDS (H-PGDS) localized in mast cells (1), Th2 cells (2) and microglia (4); and the other is lipocalin-type PGDS (L-PGDS) localized in leptomeninges, choroids plexus, and oligodendrocytes in the brain (5,6). Asthmatic responses are attenuated in mice whose gene for the DP (DP1) receptor specific for PGD2 has been knocked out (7). The DP1 receptor is constitutively expressed in human basophils and eosinophils and is induced in pulmonary and airway epithelial cells by allergens and inflammation (7). PGD2 also acts as a ligand for an orphan receptor, CRTH2 (DP2), which is expressed in human Th2 cells, eosinophils, and basophils, and mediates the chemotaxis of these cells toward PGD2 (8,9). Molecular and pharmacological properties of DP1 and DP2 receptors are summarized in Table 1. In contrast, overproduction of PGD2 exacerbates asthmatic responses, as demonstrated in ovalbuminchallenged mice transgenic for human L-PGDS (10). PGD2 is further converted to 9α,11β-PGF2, a stereoisomer of PGF2α, which exerts various pharmacological actions different from those induced by PGF2α (reviewed by Smith et al. (11)). PGD2 is also readily dehydrated in vitro (12) to produce PGs of the J series, such as PGJ2, ∆-PGJ2, and 15-deoxy-∆-PGJ2. Hematopoietic prostaglandin D synthase 137 Figure 1. Prostanoid cascade. Table 1. Biochemical and pharmacological properties of DP1 and DP2 receptors. 15-Deoxy-∆-PGJ2 acts as a ligand for a nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ), and promotes adipocyte differentiation (13,14). Although the production and occurrence of the J series of PGs in vivo have long been proposed by several research groups (15), such a proposition is now extremely questionable, because we and other groups have never detected the J series of PGs in fresh tissue samples or body fluids (16,17). Yoshihiro Urade et al. 138 Biochemical properties 1) Enzymatic properties of H-PGDS H-PGDS was originally purified in 1979 from rat spleen by ChristHazelhof and Nugteren as a cytosolic monomeric glutathione (GSH)-requiring PG endoperoxide D-isomerase (EC 5.3.99.2, Fig. 2) with a Mr of 26,000 (18). Since the Mr of H-PGDS was the same as that of L-PGDS (19-21), which had previously been miss-identified as a protein with a Mr of 80,000 (22), we reexamined the biochemical characteristics of H-PGDS and confirmed that HPGDS was quite distinct from L-PGDS in terms of kinetic parameters, amino acid composition, and immunological properties (23). During the reexamination study, we found that H-PGDS was associated with the activity of GSH S-transferase (GST). Although both H-PGDS and L-PGDS catalyze the same reaction, these enzymes have evolved from their ancestral origins differently from each other, H-PGDS from GST (as described later) and L-PGDS from lipocalins, which bind and transport various lipophilic substances (20,21,24). Therefore, we proposed that H-PGDS and L-PGDS are novel examples of functional convergence (3,25). Figure 2. Enzymatic reaction of PGH2 D-isomerase. 2) Activation of human H-PGDS by Mg We recently found that Ca and Mg ions increased the activity of HPGDS to ∼150% of its basal level in a concentration-dependent manner (Fig. 3), with half-maximum effective concentrations of 400 μM for Ca and 50 μM for Mg (26). Ca did not change the affinity of human H-PGDS for GSH (Km = 0.60 and 0.59 mM in the absence or presence, respectively, of 2 mM CaCl2); whereas Mg increased the affinity for GSH, decreasing the Km value 4-fold to 0.14 mM. Although at the highest soluble concentration of PGH2 (0.4 mM) the PGDS activity was not saturated, the calculated Km value for PGH2 changed slightly, from 0.46 mM in the absence of Ca or Mg to 0.33 or 0.93 mM in the presence of 2 mM CaCl2 or MgCl2, respectively. HPGDS is localized in the cytosol, where the concentration of Mg is estimated to be on the order of several mM. Thus, H-PGDS likely exists as the Mgbound form in vivo. Hematopoietic prostaglandin D synthase 139 Figure 3. Activation of human H-PGDS by Mg and Ca (26). 3) Homodimer of H-PGDS Although H-PGDS has been described as a monomeric protein (18,23), we recently demonstrated that H-PGDS is actually a homodimeric protein. Ultracentrifugation analysis revealed both rat and human H-PGDS's to be homodimers with a Mr of 45,000 to 49,000 in the presence or absence of Ca or Mg (Table 2). Table 2. Mr value determined by ultracentrifugation analysis. 4) Inhibition of H-PGDS by HQL-79 We recently demonstrated that the orally active anti-allergic drug 4benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]-piperidine (HQL-79) is a specific inhibitor of human H-PGDS (27). Although HQL-79 was developed as an antagonist for histamine H1 receptors, a part of the anti-allergic and antiasthmatic effects of HQL-79 was proposed to be mediated by the inhibition of PGD2 production, because HQL-79 inhibited the conversion of PGH2 to PGD2 in crude extracts of mouse spleen (28). HQL-79 inhibited the activity of purified recombinant human H-PGDS with an IC50 of 6 μM, but had almost no effect on the activities of purified COX-1, COX-2, L-PGDS or microsomal Yoshihiro Urade et al. 140 PGE synthase-1 (mPGES-1) when used up to 300 μM (Fig. 4). As described above, Mg activates human H-PGDS about 2-fold and increases its affinity for GSH about 4-fold. In the absence of Mg, the IC50 value of HQL-79 was increased about 3-fold, from 6 μM to 16 μM. Kinetic analysis revealed that HQL-79 inhibited the H-PGDS activity in a competitive manner against PGH2 (Fig. 5A), giving a Ki of 5 μM, and in a non-competitive one against GSH (Fig. 5B) with a Ki of 3 μM, in the presence of 1 mM MgCl2. These results indicate that HQL-79 bound to the PGH2binding site but not to the GSH-binding site. Figure 4. Selective inhibition of H-PGDS by HQL-79 (27). Figure 5. Kinetic analysis of inhibition of H-PGDS by HQL-79 (27). 5) Binding of HQL-79 to H-PGDS Surface plasmon resonance (SPR) analysis showed that HQL-79 bound to H-PGDS in a concentration-dependent, saturable, and Mgand GSHaccelerated manner and dissociated from the enzyme-inhibitor complex immediately when washed (Fig. 6A). In the presence of 2 mM MgCl2 and 2 mM Hematopoietic prostaglandin D synthase 141 Figure 6. SPR analysis of binding of HQL-79 to H-PGDS (27). GSH, HQL-79 bound to human H-PGDS in a concentration-dependent manner, with almost complete saturation up to 25 μM (Fig. 6B). From the association and dissociation curves, the Kd for HQL-79 was calculated to be 0.8 μM. In the absence of MgCl2 and in the presence of 2 mM GSH, the HQL79 binding to human H-PGDS significantly decreased, showing saturation at 50 μM and a Kd of 5 μM, indicating that the affinity of H-PGDS for HQL-79 decreased 6-fold in the absence of MgCl2. In the absence of GSH, the HQL-79 binding decreased the total capacity to about 50% and increased the Kd to 11 μM in the presence of MgCl2 and to 10 μM in its absence. When we determined the GSH-dependency of the HQL-79-binding to human H-PGDS in the presence of MgCl2, the binding affinity increased in a GSH concentration-dependent manner (Fig. 6C). The half-effective concentration of GSH for an increase in the affinity for HQL-79 and a decrease in the Kd was calculated to be 0.09 mM (Fig. 6D), which is similar to the Km of the H-PGDS activity for GSH (0.14 mM), suggesting that GSH binding to the catalytic site of H-PGDS was involved in the increase in the binding affinity for HQL-79. Yoshihiro Urade et al. 142 Rat H-PGDS showed an HQL-79-binding curve with a Kd of 0.7 μM. Similar to the human enzyme, in the absence of MgCl2 the rat H-PGDS showed a 5-fold decrease in the binding affinity for HQL-79, giving the Kd value of 3.4 μM, without changing the maximum binding capacity. In the absence of GSH, the rat enzyme decreased the total binding capacity to about 50% and increased the Kd value to 22 μM in the presence of MgCl2 and to 21 μM in its absence (Fig. 6E). 6) Functional coupling between H-PGDS and COX HQL-79 inhibited either antigenor Ca-ionophore (A23187)-induced production of PGD2 from [1-C] arachidonic acid in rat mastocytoma RBL2H3 cells (Fig. 7) and human megakaryocytes in a concentration (3-300 μM)dependent manner, both of which express predominantly H-PGDS. However, the production of other [C]-labeled metabolites was not inhibited by HQL-79 used up to 300 μM. This effect was quite different from that of indomethacin, which inhibited the production of all PGs. Moreover, HQL-79 had no effect on the production of PGD2 by L-PGDS-over-expressing HEK-293 cells or human TE-671 cells (27), both of which predominantly express L-PGDS. Figure 7. Selective inhibition of production of C-PGD2 in RBL cells by HQL-79 (27). AA, arachidonic acid; HHT, 12-hydroxyheptadecatrienoic acid; and Ind, indomethacin. The IC50 value of HQL-79 for inhibition of PGD2 production in megakaryocytes was calculated by EIA to be 102 μM (Fig. 8). HQL-79 at a concentration of 300 μM decreased PGD2 production to 3.1 ng/10 cells from 10.1 ng/10 vehicle-treated cells; whereas it increased PGE2 production to 0.32 ng/10 cells from 0.17 ng/10 vehicle-treated cells and decreased PGF2α production to 0.23 ng/10 cells from 0.34 ng/10 vehicle-treated cells. HQL-79 tested up to 300 μM did not affect at all the production of PGD2, PGE2 or PGF2α in the L-PGDS-over-expressing HEK-293 cells. Hematopoietic prostaglandin D synthase 143 Figure 8. Selective inhibition of production of PGD2 in megakaryocytes by HQL-79 (27). Selective inhibition by HQL-79 of PGD2 accumulated in the culture medium of MEG-01S cells. The amounts of PGD2, PGE2, and PGF2α were measured by EIA. Data represent the mean ± SEM (n=4). *p<0.05, **p<0.01 as compared with the value in the absence of HQL-79. ††p<0.01 as compared with the value in the presence of 300 μM HQL-79 (Dunnett’s test). These results indicate that the inhibition of H-PGDS decreased PGD2 production selectively without significantly affecting the biosynthesis of other PGs. Once the downstream H-PGDS was inhibited, the upstream COX was also inhibited, suggesting that H-PGDS and COX were functionally tightly engaged with each other (Fig. 9). In this sense, HQL-79 is an even better PG-blocking compound than those available today (29). Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used as anti-inflammatory drugs that ameliorate pain, fever, and inflammation by blocking PG production. However, NSAIDs inhibit the production of all prostanoids, including the cytoprotective and anti-inflammatory PGs (Fig. 9). For example, aspirin and indomethacin induce gastrointestinal toxicity by blocking PGE2 production. The anti-inflammatory action of PGE2 mediated by EP3 receptors was also very recently reported (30). We previously demonstrated that PGD2 produced by L-PGDS prevents neuronal and oligodendroglial apoptosis during neuroinflammation in a genetic demyelination mouse model, i.e., twitcher (31). Thus, HQL-79 may be predicted to selectively suppress the inflammatory reaction mediated by H-PGDS-catalyzed PGD2 without various side effects caused by the suppression of cytoprotective and anti-inflammatory PGs. Yoshihiro Urade et al. 144 Figure 9. Comparison of selective inhibition of PGD2 production by HQL-79 with total inhibition of PG production by Ind. indomethacin. 7) Tissue and cellular distribution of H-PGDS Immunoabsorption analysis of the PGDS activity in various rat tissues with anti-H-PGDS antibodies revealed that H-PGDS contributes to the production of PGD2 in the spleen, thymus, intestine, and various peripheral tissues of rats (32). Northern blot analysis showed that the tissue distribution profile of the mRNA for H-PGDS highly deviated among various species including rats (33), humans, mice (34), and chickens (35). However, H-PGDS was highly expressed in the oviduct of all 3 mammalian species, indicating that H-PGDS plays an important role in the female genital organ. H-PGDS is immunohistochemically or immunocytochemically localized in Langerhans cells in the skin (36), Kupffer cells in the liver, dendritic cells in the thymus and intestine (37), mast cells (1) of variety of rat and human tissues; human megakaryocytes (38), activated Th2 cells (2), and eosinophils (39); microglia of the mouse brain (4,41); and necrotic muscle of mdx mice (I.M., Y.U.; unpublished results) or patients with Duchenne's muscular dystrophy or polymyositis (41). Evolutional properties of H-PGDS We have already obtained the full-length cDNA for rat H-PGDS (33) followed by that for the human and mouse enzymes (34). The cDNA encodes a Hematopoietic prostaglandin D synthase 145 protein composed of 199 amino acid residues with a calculated Mr of 23,297, 23,343, and 23,226 for the rat, human, and mouse enzymes, respectively. The first N-terminal methionine is cleaved from the mature enzyme. The cDNA for the chick homolog was isolated by Thompson et al. (35). A database search of the protein primary structure revealed H-PGDS to be a member of the GST family, as predicted by the results obtained by partial amino acid sequence analysis. The amino acid sequence of H-PGDS was similar to those of all GST isozymes from the previously known 5 classes: alpha, mu, pi, sigma, and theta (Fig. 10), showing weak similarity (<30% identity) to mammalian GST isozymes, yet moderate similarity (32% to 40% identity) to the insect and fluke GST, and the highest identity to GSTs of the house fly (Musca domestica, 40% identity) and pig roundworm (Ascaris suum, 39% identity) in the multiple sequence alignment. In a phylogenetic tree, H-PGDS formed a subcluster with members of the sigma class GST including S-crystallins from cephalopods (Fig. 11). The sigma class GSTs were previously reported to be present in invertebrates such as insects, cephalopods, flukes, and nematodes. Thus, H-PGDS is a novel vertebrate homolog of the sigma class of the GST family. Among members of the sigma-class GST family, H-PGDS is the most related to C. elegans GST (σ). Members of the alpha, mu, and pi-class GSTs and squid GST (σ) of the sigma-class GST have the ability to convert PGH2 to a mixture of PGD2, PGE2, and PGF2α in the presence of GSH (42,43). However, their PGD synthase activity was lower than their PGE synthase and PGF synthase activities. Therefore, we propose that the H-PGDS gene evolved from a common ancestor of the Figure 10. Homology of H-PGDS with members of GST family (34). Yoshihiro Urade et al. 146 Figure 11. Phylogenetic tree of H-PGDS and GSTs (34). invertebrate sigma-class GSTs, and acquired specifically PGD synthase activity during its evolution. Metal activation of the enzyme activity was not observed in any other GST isozymes in the alpha, mu, pi, and sigma classes or in the sigma class GST from Schistosoma mansoni, thus indicating that the metal activation is specific to H-PGDS among the GST family. H-PGDS is a unique Mg-containing GST. Crystallographic structure of H-PGDS We have determined the X-ray crystallographic structures of H-PGDS of 4 distinct complexes, as summarized in Table 3 (26, 27, 34). Table 3. Summary of crystallographic structures of 4 distinct complexes of H-PGDS. Hematopoietic prostaglandin D synthase 147 1) Rat enzyme as a binary complex with GSH We first determined the crystal structure of the rat H-PGDS.GSH complex at 2.3 Å resolution by the multiple isomorphous replacement method (33). The recombinant rat H-PGDS was crystallized in a trigonal P3122 form. The crystal was obtained as a homodimer in an asymmetric unit, and each monomer was complexed with 1 GSH molecule (Fig. 12). The monomers were related by a non-crystallographic 2-fold axis. The dimer interaction showed the "lock-andkey" complimentarity feature of a hydrophobic surface of Phe48 with a limited number of the electrostatic interactions (Fig. 12), which is commonly observed in various GSTs. The dimensions of the H-PGDS monomer are about 50 x 50 x 30 Å, and the overall folding motif is the same as those of GSTs. The monomer is constructed from 2 domains with a prominent interdomain cleft (Fig. 12); the N-terminal domain (amino acid residues 1–71) and the C-terminal domain (82–199) are connected by the residues 72–81 including 2 turn structures (72–75 and 76–79). The N-terminal domain contains a 4-stranded β sheet and 3 α helices, arranged in a βαβαββα motif, in which the β1 and β2 are parallel and the β1 and β3, and the β3 and β4, are antiparallel. The α1 and α3 helices make the dimer interface with the α4, α6, and α8 of the counterpart in the dimer. The loop structure (46–52) bends at the position of Pro52 to the outside of the enzyme, which is the GSH binding site. The angle between the directions of the loop and the β3 backbone is approximately 90°, so that the side chains of the loop residues are exposed to the solvent with the Ile51 residue in cis-conformation, resulting in the formation of a GSH-binding dimple. The C-terminal domain is composed of 5 α helices, in which the α4, α5, and α6 make an α-helix bundle. The long α5 bends at the position of Gln123. In the connecting loop of the α4 and α5 helices, the backbone between Ser100 and Trp104 is kinked to compensate for the α4, which is shorter than those of other GSTs in the amino acid alignment. Figure 12. Crystallographic structure of rat H-PGDS as a binary complex with GSH (33). Yoshihiro Urade et al. 148 GSH is bound to the side of the N-terminal domain (Fig. 13) by forming 2 and 8 hydrogen bonds to the atoms of the protein backbone and side chain, respectively, similar to other GSTs. The cysteinyl backbone of GSH interacts with that of cis-Ile51 of the N-terminal domain via hydrogen bonds in an antiparallel β-sheet manner. The amino nitrogen of the γ-glutamate residue of GSH forms hydrogen bonds with both of the carboxyl oxygens of the Asp97 side chain of the other H-PGDS molecule in the dimer. The Sγ atom of GSH and the Oη of Tyr8 show a hydrogen bonding distance of 3.1 Å. All these residues are highly conserved among the members of the GST family. The prominent cleft including the GSH binding site between the 2 domains is the catalytic pocket. The interdomain cleft expands to a wide and deep pocket (pocket 1) behind the GSH binding site with the longest loop eaves of the α4 and α5 helices. At the entrance of pocket 1, the indole ring of Trp104 forms a ceiling on the C-terminal domain, since the indole ring is directed parallel to the α4 helix extended by the kinked backbone including Trp104. Pocket 1 has a path to a branched cavity consisting of another pocket (pocket 2) and a narrow tunnel. Pocket 1 also opens to a third pocket (pocket 3) on the outer surface due to the short C-terminal end of H-PGDS. There is a straight path from the outside of the protein to pocket 2 via pockets 3 and 1. As for the dimensions of the cavities, pocket 1 is 5 Å in depth by 6 Å in width and opens to the GSH binding site. Pocket 2 is 4 Å in depth and 6 Å in width and is lined with hydrophilic residues in contrast to pockets 1 and 3, whose surfaces are hydrophobic with many aromatic side chains. In the bottom of pocket 2, there are 2 bound water molecules that are 2.9 Å apart; and they form hydrogen bonds with the 0η of Tyr152 and the Nζ of Arg14, which are at the distances of 8.7 Å and 8.1 Å, respectively, from the Sγ atom of GSH. The electrostatic potential on the surface within the cleft including GSH is positive around the guanidino group of Arg14, negative along GSH, and neutral in the other part. Figure 13. GSH-binding to the catalytic cleft of rat H-PGDS (33). Hematopoietic prostaglandin D synthase 149 2) Human H-PGDS as the ternary complex with GSH and Ca or Mg We then obtained monoclinic P21 crystals of human H-PGDS in the presence of Ca and GSH, and determined the structure at 1.8 Å resolution (26). The asymmetric unit of the crystal lattice contained 2 enzyme-dimers, designated Mol-A/Mol-D and Mol-C/Mol-B (Fig. 14A). The human H-PGDS dimer structure (Fig. 14B) was identical to that in the trigonal crystal of rat HPGDS (Fig. 12), and the overall fold of each H-PGDS subunit was essentially the same as that of other GSTs and rat H-PGDS. The subunits in the dimers had similar overall folds: the r.m.s. deviations were 0.23 Å for Mol-A/Mol-B, 0.42 Å for Mol-A/ Mol-C, 0.29 Å for MolA/Mol-D, 0.43 Å for Mol-B/Mol-C, 0.30 Å for Mol-B/Mol-D, and 0.35 Å for Mol-C/Mol-D for all Cα atoms except for the region of helix α5. Specifically, Tyr122 in helix α5 of Mol-A and Mol-B was clearly distinct from that of MolC and Mol-D; Tyr122 of Mol-A and Mol-B but not that of Mol-C and Mol-D could make a hydrogen bond with His87 in the neighboring dimers in the crystal. Moreover, the structure of the glycine residue of the bound GSH in Mol-B was clearly different from that of Mol-A, Mol-C, and Mol-D. The high-resolution structure revealed Ca to be at the center of the dimer interface (Fig. 14B). The Ca-binding site consisted of pairs of Asp93, Asp96, and Asp97 from each monomer. These 6 aspartates formed an acidic cluster at the dimer interface, and the residues from each pair were related via a noncrystallographic two-fold symmetry axis at the center of the dimer. The Cabinding site was at a hinge portion between the Nand C-terminal domains of the subunit (Fig. 14C). The Ca ion was directly coordinated by 5 water molecules (W1–W3, W5, and W6) and Asp96 in Mol-C or Mol-D (Fig. 15). Asp96 from Mol-A or Mol-B interacted with the coordinated water molecule W6; and Asp93 and Asp97, with the Ca ion through water molecules (W5 and W1 and W2, respectively). The distance between O1(Asp96) of Mol-C or Mol-D and Ca was 2.8 Å; whereas that between Ca and W6 was 2.0 Å, and that between W6 and O1(Asp96) of Mol-A (or Mol-B) was 2.5 Å. In the dimer, each of the O2 (Asp96) atoms made 2 hydrogen bonds with the guanidium nitrogen atoms of Arg14 in the same subunit. In turn, each of the N2 (Arg14) atoms formed a hydrogen bond with an Oγ(Ser100) in the same subunit, but the observed distances were different: 3.0 Å for Mol-A or Mol-B and 2.6 Å for Mol-C or Mol-D. The Arg14 residue was involved in the activation of the thiol of GSH and in recognition of the ω-chain of the substrate PGH2. Thus, Ca, Asp96, Arg14, and Ser100 formed a hydrogenbonding network at the active site of human H-PGDS. The structure of human H-PGDS in the presence of Mg was also determined, at 1.7 Å resolution (26). The space group of the crystal and the Yoshihiro Urade et al. 150 Figure 14. Crystallographic structure of human H-PGDS as ternary complexes with GSH and Ca (26). Figure 15. The metal coordination structures of human H-PGDS (A), Mol-A; (D),