The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H 2 from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co 3؉ -protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 Å , respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.The cyclooxygenase enzymes (COX-1 and COX-2) are membrane-associated heme-containing bifunctional enzymes that catalyze the first committed step in prostaglandin (PG) 2 biosynthesis (reviewed in Refs. 1, 2). The product, PGH 2 , is produced as a result of two sequential reactions that are performed in separate but functionally linked active sites. In the first reaction, arachidonic acid (AA; 20:4 -6) bound in the cyclooxygenase channel undergoes a bis-oxygenation to form the intermediate PGG 2 . The released intermediate is then bound in the peroxidase active site, where the 15-hydroperoxide group of PGG 2 is reduced to form PGH 2 in the second reaction. Both COX-1 and COX-2 require a preliminary catalytic turnover at the peroxidase active site to generate an oxy-ferryl porphyrin radical that is subsequently transferred to Tyr-385 for the initiation of cyclooxygenase catalysis.Understanding the similarities and differences associated with the structure and function of COX-1 and COX-2 and the rationale for the existence of two isoforms has been the focus of much recent research (3-5). COX-1 and COX-2, which are encoded by separate genes (6), display ϳ60% sequence identity within the same species and greater than 85% sequence identity among orthologs from different species (7). Accordingly, the crystal structures of COX-1 and COX-2 are virtually superimposable, and the catalytic mechanism is conserved between isoform...

Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H 2 . Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E cat ) and an allosteric monomer (E allo ). Heme binds tightly only to the peroxidase site of E cat , whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E cat . E cat is regulated by E allo in a manner dependent on what ligand is bound to E allo . Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E allo . AA can bind to E cat and E allo , but the affinity of AA for E allo is 25 times that for E cat . Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E allo in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E cat or E allo . Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both baseline prostanoid synthesis and responses to COX inhibitors.Prostaglandin endoperoxide H synthases (PGHSs), 2 also known generically as cyclooxygenases (COXs), convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step of prostanoid biosynthesis (1-6). PGHS-1 and PGHS-2 are products of different genes. In general, PGHS-1 is constitutively expressed, whereas PGHS-2 expression is inducible (4, 5, 7). The enzymes are embedded in the luminal monolayer of the endoplasmic reticulum and inner membrane of the nuclear envelope (8 -11).PGHSs catalyze two different reactions, a COX reaction and a peroxidase (POX) reaction. COX catalysis begins with abstraction of the 13-pro-S-hydrogen from AA by the enzyme in the rate-determining step to generate an arachidonyl radical (3,6,12,13). Two molecules of O 2 are then sequentially added to the arachidonyl chain with concomitant rearrangements to form PGG 2 . PGG 2 can be reduced to PGH 2 by the POX activity. The purified isoforms are about equally efficient in catalyzing the conversion of AA to PGH 2 (1-6).PGHSs are homodimers composed of tightly associated monomers with identical sequences (14). Each monomer comprising a PGHS homodimer has a physically distinct COX and POX active site. Dissociation of the dimers into monomers only occurs upon denaturation (14). Kulmacz and Lands (15,16) provided the first evidence that the monomers of PGHS homodimers differ. They found that maximal COX activity of...

Aspirin and other nonsterroidal anti-inflammatory drugs target the Cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the mono-oxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for oxygen addition into the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9Å, 2.0Å, and 2.4Å, respectively. The structures reveal that: 1) the acetylated Ser-530 completely blocks access to the hydrophobic groove; 2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation; and 3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove. Based on these structural observations, along with functional analysis of the S530T/G533V double mutant, we propose a working hypothesis for the generation of 15R-HETE by aspirin-acetylated COX-2. We also observe differential acetylation of COX-2 purified in various detergent systems and nanodiscs, indicating that detergent and lipid binding within the membrane-binding domain of the enzyme alters the rate of the acetylation reaction in vitro.

The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 Å , respectively, and R513H muCOX-2 in complex with AA to 2.45 Å resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.The reaction catalyzed by the cyclooxygenase enzymes (COX-1 and COX-2), 2 in which prostaglandin H 2 (PGH 2 ) is generated from the fatty acid substrate arachidonic acid (AA), represents the committed step in the biosynthesis of prostaglandins and thromboxanes (reviewed in Refs. 1 and 2). Downstream synthases subsequently convert PGH 2 into potent lipid-signaling molecules that are tasked with regulating "housekeeping" functions required for normal physiological activities and that are also associated with a number of pathologies, including inflammation and cancer (3, 4). Both COX-1 and COX-2 are the pharmacological targets of aspirin and other nonsteroidal anti-inflammatory drugs, which are utilized for the treatment of pain and inflammation (5). COX-2 can also be selectively inhibited by the recently developed coxibs: diarylheterocycle-based compounds that include celecoxib, rofecoxib, valdecoxib, and etoricoxib (6). The catalytic mechanism and molecular basis underlying the conversion of AA to PGH 2 by COX-1 and COX-2 has been established utilizing structural and functional studies carried out over the past 20ϩ years (reviewed in Refs. 7 and 8). The COX enzymes are membrane-associated, heme-containing homodimers that contain spatially distinct but functionally linked cyclooxygenase and peroxidase active sites. In its productive conformation, AA is oriented in the cyclooxygenase channel with th...

The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9–expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.

Background: Cyclooxygenase-1 and -2 have isoform-specific substrate preferences and interactions. Results: Mutations to residues in the COX-2 cyclooxygenase channel individually have no significant effect on high affinity binding of fatty acid substrates. Conclusion: Proper bisallylic carbon positioning drives efficient substrate oxygenation by COX-2. Significance: Our results confirm nuances between isoforms that explain the ability of COX-2 to oxygenate a broad spectrum of substrates.

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell–cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers—but the basis for CpE’s selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin–enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin–CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

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