Defining the Conformational Ensembles Associated with Ligand Binding to Cyclooxygenase-2.

Cyclooxygenases (COX) catalyze the committed step in the production of prostaglandins responsible for the maintenance of physiological homeostasis. While crystal structures of COX in complex with substrates and inhibitors have provided insight into the molecular interactions governing their binding, they have not uncovered specific details related to the protein conformational motions responsible for important aspects of the COX function. We created a cysteine-free COX-2 construct and introduced a free cysteine at position-122 to enable labeling with 3-bromo-1,1,1-trifluoroacetone (BTFA).

Functional and structural analyses reveal that a dual domain sialidase protects bacteria from complement killing through desialylation of complement factors.

The complement system is the first line of innate immune defense against microbial infections. To survive in humans and cause infections, bacterial pathogens have developed sophisticated mechanisms to subvert the complement-mediated bactericidal activity. There are reports that sialidases, also known as neuraminidases, are implicated in bacterial complement resistance; however, its underlying molecular mechanism remains elusive.

Anti-σ Factor FlgM Regulates Flagellin Gene Expression and Flagellar Polarity of Treponema denticola.

FlgM, an antagonist of FliA (also known as σ), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliA) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized.

Identification of a homozygous recessive variant in resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes.

An Activity-Based Sensing Approach for the Detection of Cyclooxygenase-2 in Live Cells.

Cyclooxygenase-2 (COX-2) overexpression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX-2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity-based sensing approach for monitoring COX-2 within live cells with confocal microscopy and flow cytometry.

Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.

Aspirin and Celebrex are well-known time-dependent inhibitors of the cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex.

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi.

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook-associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi.

Interactions of fatty acids, nonsteroidal anti-inflammatory drugs, and coxibs with the catalytic and allosteric subunits of cyclooxygenases-1 and -2.

Prostaglandin endoperoxide H synthases-1 and -2, commonly called cyclooxygenases-1 and -2 (COX-1 and -2), catalyze the committed step in prostaglandin biosynthesis-the conversion of arachidonic acid to prostaglandin endoperoxide H Both COX isoforms are sequence homodimers that function as conformational heterodimers having allosteric (Eallo) and catalytic (Ecat) subunits. At least in the case of COX-2, the enzyme becomes folded into a stable Eallo/Ecat pair. Some COX inhibitors ( nonsteroidal anti-inflammatory drugs and coxibs) and common fatty acids (FAs) modulate Ecat activity by binding Eallo.

Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.

Aspirin (acetylsalicylic acid) inhibits prostaglandin (PG) synthesis by transfer of its acetyl group to a serine residue in the cyclooxygenase (COX) active site. Acetylation of Ser530 inhibits catalysis by preventing access of arachidonic acid substrate in the COX-1 isoenzyme. Acetylated COX-2, in contrast, gains a new catalytic activity and forms 15 R hydroxy-eicosatetraenoic acid (15 R-HETE) as alternate product.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge.

A Cation-Binding Surface Protein as a Vaccine Antigen To Prevent Moraxella catarrhalis Otitis Media and Infections in Chronic Obstructive Pulmonary Disease.

is an exclusively human respiratory tract pathogen that is a common cause of otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent these infections would have a major impact on reducing the substantial global morbidity and mortality in these populations. Through a genome mining approach, we identified AfeA, an ∼32-kDa substrate binding protein of an ABC transport system, as an excellent candidate vaccine antigen.

Fatty Acid Binding to the Allosteric Subunit of Cyclooxygenase-2 Relieves a Tonic Inhibition of the Catalytic Subunit.

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH PGHS-2 is a conformational heterodimer composed of allosteric (E) and catalytic (E) subunits. Fatty acids (FAs) bind to Arg-120 of E increasing to different degrees, depending on the FA, the V of its E partner. We report here that movement of helical residues 120-122 and loop residues 123-129 of E underlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen.

Crystal structure of rofecoxib bound to human cyclooxygenase-2.

Rofecoxib (Vioxx) was one of the first selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) to be approved for use in humans. Within five years after its release to the public, Vioxx was withdrawn from the market owing to the adverse cardiovascular effects of the drug. Despite the widespread knowledge of the development and withdrawal of Vioxx, relatively little is known at the molecular level about how the inhibitor binds to COX-2.

Sulfate-binding protein, CysP, is a candidate vaccine antigen of Moraxella catarrhalis.

Moraxella catarrhalis causes otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). A vaccine to prevent M. catarrhalis infections would have an enormous impact globally in preventing morbidity caused by M.

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid.

Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry.

Aspirin and other nonsteroidal 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 monooxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE).

Pulsed Dipolar Spectroscopy Reveals That Tyrosyl Radicals Are Generated in Both Monomers of the Cyclooxygenase-2 Dimer.

Cyclooxygenases (COXs) are heme-containing sequence homodimers that utilize tyrosyl radical-based catalysis to oxygenate substrates. Tyrosyl radicals are formed from a single turnover of substrate in the peroxidase active site generating an oxy-ferryl porphyrin cation radical intermediate that subsequently gives rise to a Tyr-385 radical in the cyclooxygenase active site and a Tyr-504 radical nearby. We have utilized double-quantum coherence (DQC) spectroscopy to determine the distance distributions between Tyr-385 and Tyr-504 radicals in COX-2.

The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis.

Moraxella catarrhalis is an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent M. catarrhalis infections would have an enormous global impact in reducing morbidity resulting from these infections.

The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase.

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H).

Structural characterization and modeling of the Borrelia burgdorferi hybrid histidine kinase Hk1 periplasmic sensor: A system for sensing small molecules associated with tick feeding.

Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks.

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T.

The structure of ibuprofen bound to cyclooxygenase-2.

The cyclooxygenases (COX-1 and COX-2) catalyze the rate-limiting step in the biosynthesis of prostaglandins, and are the pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). Ibuprofen (IBP) is one of the most commonly available over-the-counter pharmaceuticals in the world. The anti-inflammatory and analgesic properties of IBP are thought to arise from inhibition of COX-2 rather than COX-1.

Role of the oligopeptide permease ABC Transporter of Moraxella catarrhalis in nutrient acquisition and persistence in the respiratory tract.

Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival.

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment.

Crystal structures of α-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

α-Dioxygenases (α-DOX) are heme-containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α-DOX oxygenate a variety of 14-20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro-R hydrogen from the α-carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H2 O2 ). We determined the X-ray crystal structures of wild type α-DOX from Oryza sativa, the wild type enzyme in complex with H2 O2 , and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA).