Masashi Miyano

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

BACKGROUND Hepatitis C virus (HCV) is the major etiological agent of hepatocellular carcinoma, and HCV RNA-dependent RNA polymerase (RdRp) is one of the main potential targets for anti-HCV agents. HCV RdRp performs run-off copying replication in an RNA-selective manner for the template-primer duplex and the substrate, but the structural basis of this reaction mechanism has still to be elucidated. RESULTS The three-dimensional structure of HCV RdRp was determined by X-ray crystallography at 2.5 A resolution. The compact HCV RdRp structure resembles a right hand, but has more complicated fingers and thumb domains than those of the other known polymerases, with a novel alpha-helix-rich subdomain (alpha fingers) as an addition to the fingers domain. The other fingers subdomain (beta fingers) is folded in the same manner as the fingers domain of human immunodeficiency virus (HIV) reverse transcriptase (RT), another RNA-dependent polymerase. The ribose-recognition site of HCV RdRp is constructed of hydrophilic residues, unlike those of DNA polymerases. The C-terminal region of HCV RdRp occupies the putative RNA-duplex-binding cleft. CONCLUSIONS The structural basis of the RNA selectivity of HCV RdRp was elucidated from its crystal structure. The putative substrate-binding site with a shallow hydrophilic cavity should have ribonucleoside triphosphate (rNTP) as the preferred substrate. We propose that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps. The C-terminal region might exert a regulatory function on the initiation and activity of HCV RdRp.

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 Å resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane α-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

Crystal Structure of Squid Rhodopsin with Intracellularly Extended Cytoplasmic Region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7Å resolution, which transduces signals through the Gq protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to Gt, and humanβ2-adrenergic receptor, coupling to Gs. Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and humanβ2-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic α-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.

Structural and Functional Characterization of HQL-79, an Orally Selective Inhibitor of Human Hematopoietic Prostaglandin D Synthase.

We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-Å resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg2+. HQL-79 inhibited human H-PGDS competitively against the substrate PGH2 and non-competitively against GSH with Ki of 5 and 3 μm, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg2+ (Kd, 0.8 μm) than in their absence. Mutational studies revealed that Arg14 was important for the Mg2+-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD2 production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 μm but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD2, without affecting the production of PGE2 and PGF2α, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically.

CDNA CLONING, EXPRESSION, AND MUTAGENESIS STUDY OF LIVER-TYPE PROSTAGLANDIN F SYNTHASE

Prostaglandin (PG) F synthase catalyzes the reduction of PGD2 to 9α,11β-PGF2 and that of PGH2 to PGF2α on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme (K m value of 120 μm for PGD2 (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O. (1985) J. Biol. Chem. 260, 7035–7041) and the liver-type one (K m value of 10 μm for PGD2 (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992)Arch. Biochem. Biophys. 296, 17–26)). The liver-type enzyme was presently found to consist of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with aM r of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase (DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid sequence of the liver-type PGF synthase was identical with that of bovine liver DD3. The liver-type PGF synthase was expressed in COS-7 cells, and its recombinant enzyme had almost the same properties as the native enzyme. Furthermore, to investigate the nature of catalysis and/or substrate binding of PGF synthase, we constructed and characterized various mutant enzymes as follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. Although the reductase activities toward PGH2 and phenanthrenequinone (PQ) of almost all mutants were not inactivated, the K m values of R27E, R91Q, H170C, R223L, and N306Y for PGD2 were increased from 15 to 110, 145, 75, 180, and 100 μm, respectively, indicating that Arg27, Arg91, His170, Arg223, and Asn306 are essential to give a low K m value for PGD2 of the liver-type PGF synthase and that these amino acid residues serve in the binding of PGD2. Moreover, the R223L mutant among these seven mutants especially has a profound effect on k cat for PGD2 reduction. TheK m values of R223L, K225S, and S301R for PQ were about 2–10-fold lower than the wild-type value, indicating that the amino acid residues at 223, 225 and 301 serve in the binding of PQ to the enzyme. On the other hand, the K m value of H170C for PGH2 was 8-fold lower than that of the wild type, indicating that the amino acid residue at 170 is related to the binding of PGH2 to the enzyme and that Cys170 confer high affinity for PGH2. Additionally, the 5-fold increase in k cat/K m value of the N306Y mutant for PGH2 compared with the wild-type value suggests that the amino acid at 306 plays an important role in catalytic efficiency for PGH2.

Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order Co2+ ≥ Mn2+ ≥ Mg2+ » Ca2+ ≥ Sr2+. The first crystal structures of Bc-SMase bound to Co2+, Mg2+, or Ca2+ were determined. The water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg2+ or Co2+ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific β-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.

Structural Basis of Hematopoietic Prostaglandin D Synthase Activity Elucidated by Site-directed Mutagenesis

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH2 to PGD2 and conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. We introduced site-directed mutations of Tyr8, Arg14, Trp104, Lys112, Tyr152, Cys156, Lys198, and Leu199, which are presumed to participate in catalysis or PGH2 substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K m for glutathione and PGH2 in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K m for glutathione was increased. Using 5,5′-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys112, Cys156, and Lys198 are involved in the binding of PGH2; Trp104 is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr8 and Arg14 are essential for activation of the thiol group of glutathione.

Mechanism of metal activation of human hematopoietic prostaglandin D synthase.

Here we report the crystal structures of human hematopoietic prostaglandin (PG) D synthase bound to glutathione (GSH) and Ca2+ or Mg2+. Using GSH as a cofactor, prostaglandin D synthase catalyzes the isomerization of PGH2 to PGD2, a mediator for allergy response. The enzyme is a homodimer, and Ca2+ or Mg2+ increases its activity to ∼150% of the basal level, with half maximum effective concentrations of 400 μM for Ca2+ and 50 μM for Mg2+. In the Mg2+-bound form, the ion is octahedrally coordinated by six water molecules at the dimer interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca2+ is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca2+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg2+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the Km of the enzyme for GSH. The structure provides insights into how Ca2+ or Mg2+ binding activates human hematopoietic PGD synthase.

Helix 8 of the Leukotriene B4 Receptor Is Required for the Conformational Change to the Low Affinity State after G-protein Activation

Recent studies have revealed that G-protein-coupled receptors contain a putative cytoplasmic helical domain, helix 8. Leukotriene B4 (LTB4) receptor 1 derivatives with truncated or mutated helix 8 showed much higher LTB4 binding than wild-type (WT) receptors. Similar to the WT receptor, LTB4 promoted guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding in these mutants. Unlike the WT receptor, however, the addition of GTPγS did not inhibit LTB4 binding to the mutant receptors. Scatchard analyses revealed that mutants maintained high affinity for LTB4, even in the presence of excess GTPγS. Consistently, mutant receptors showed a more prolonged Ca2+ mobilization and cellular metabolic activation than the WT receptor. From mutational studies and three-dimensional modeling based on the structure of bovine rhodopsin, we conclude that the helix 8 of LTB4 receptor 1 plays an important role in the conformational change of the receptor to the low affinity state after G-protein activation, possibly by sensing the status of coupling Gα subunits as GTP-bound.