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1.
PLoS Pathog ; 19(9): e1011674, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37747935

RESUMO

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. Several complement proteins (e.g., C1q, C4, and C5) and regulators (e.g., factor H and C4bp) are modified by various sialoglycans (glycans with terminal sialic acids), which are essential for their functions. This report provides both functional and structural evidence that bacterial sialidases can disarm the complement system via desialylating key complement proteins and regulators. The oral bacterium Porphyromonas gingivalis, a "keystone" pathogen of periodontitis, produces a dual domain sialidase (PG0352). Biochemical analyses reveal that PG0352 can desialylate human serum and complement factors and thus protect bacteria from serum killing. Structural analyses show that PG0352 contains a N-terminal carbohydrate-binding module (CBM) and a C-terminal sialidase domain that exhibits a canonical six-bladed ß-propeller sialidase fold with each blade composed of 3-4 antiparallel ß-strands. Follow-up functional studies show that PG0352 forms monomers and is active in a broad range of pH. While PG0352 can remove both N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), it has a higher affinity to Neu5Ac, the most abundant sialic acid in humans. Structural and functional analyses further demonstrate that the CBM binds to carbohydrates and serum glycoproteins. The results shown in this report provide new insights into understanding the role of sialidases in bacterial virulence and open a new avenue to investigate the molecular mechanisms of bacterial complement resistance.


Assuntos
Neuraminidase , Ácidos Siálicos , Humanos , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Sistema Complemento , Fatores Imunológicos , Porphyromonas gingivalis
2.
J Bacteriol ; 205(2): e0046322, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36715541

RESUMO

FlgM, an antagonist of FliA (also known as σ28), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ28 to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliATd) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliATd. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliATd and prevents it from binding to the σ28-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliATd turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria. IMPORTANCE Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors. While the structure and function of PFs have been extensively studied in spirochetes, the molecular mechanism that regulates the PFs' morphogenesis and assembly is poorly understood. In this report, FlgM, an anti-σ28 factor, is identified and functionally characterized in the oral spirochete Treponema denticola. Our results show that FlgM regulates the number and polarity of PFs via a unique mechanism. Identification of FliA and FlgM in T. denticola sets a benchmark to investigate their roles in other spirochetes.


Assuntos
Proteínas de Bactérias , Flagelina , Treponema denticola , Proteínas de Bactérias/genética , Escherichia coli/genética , Flagelos/metabolismo , Flagelina/genética , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Treponema denticola/genética
3.
Biochemistry ; 62(21): 3134-3144, 2023 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-37852627

RESUMO

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). Placement of the label adjacent to the cyclooxygenase channel entrance permitted the detection of alterations upon ligand binding. 19F-nuclear magnetic resonance spectroscopy (19F-NMR) was then used to probe the conformational ensembles arising from BTFA-labeled COX-2 constructs in the presence and absence of ligands known to allosterically activate or inhibit COX-2. 19F-NMR analyses performed in the presence of the time-dependent inhibitor flurbiprofen, as well as Arg-120, Tyr-355, and Glu-524 mutations, led to the classification of two ensembles as representing the relaxed and tightened states of the cyclooxygenase channel entrance. A third ensemble, generated in the presence of arachidonic acid and the Y355F mutant and modulated by the allosteric potentiators palmitic acid and oleic acid and the nonallosteric substrates 2-arachidonoyl glycerol ether and anandamide, was classified as being related to the allosteric regulation of COX activity. The ensemble-based insight into COX function demonstrated here complements the static information derived from crystal structure analyses, collectively providing a more detailed framework of the dynamics involved in the regulation of COX catalysis and inhibition.


Assuntos
Flurbiprofeno , Ciclo-Oxigenase 2/metabolismo , Ligantes , Ciclo-Oxigenase 1/metabolismo , Flurbiprofeno/farmacologia , Inibidores de Ciclo-Oxigenase 2/química , Ácido Araquidônico
4.
Haematologica ; 106(5): 1423-1432, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32299908

RESUMO

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. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.


Assuntos
Aspirina , Testes de Função Plaquetária , Plaquetas , Ciclo-Oxigenase 1/genética , Feminino , Humanos , Agregação Plaquetária/genética , Tromboxano A2
5.
J Biol Chem ; 294(5): 1697-1705, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710016

RESUMO

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 H2 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 (i.e. nonsteroidal anti-inflammatory drugs and coxibs) and common fatty acids (FAs) modulate Ecat activity by binding Eallo. However, the interactions and outcomes often differ between isoforms. For example, naproxen directly and completely inhibits COX-1 by binding Ecat but indirectly and incompletely inhibits COX-2 by binding Eallo. Additionally, COX-1 is allosterically inhibited up to 50% by common FAs like palmitic acid, whereas COX-2 is allosterically activated 2-fold by palmitic acid. FA binding to Eallo also affects responses to COX inhibitors. Thus, COXs are physiologically and pharmacologically regulated by the FA tone of the milieu in which each operates-COX-1 in the endoplasmic reticulum and COX-2 in the Golgi apparatus. Cross-talk between Eallo and Ecat involves a loop in Eallo immediately downstream of Arg-120. Mutational studies suggest that allosteric modulation requires a direct interaction between the carboxyl group of allosteric effectors and Arg-120 of Eallo; however, structural studies show some allosterically active FAs positioned in COX-2 in a conformation lacking an interaction with Arg-120. Thus, many details about the biological consequences of COX allosterism and how ligand binding to Eallo modulates Ecat remain to be resolved.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Ácidos Graxos/metabolismo , Inflamação/tratamento farmacológico , Domínio Catalítico , Humanos , Inflamação/enzimologia , Inflamação/patologia
6.
Mol Microbiol ; 111(6): 1652-1670, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30883947

RESUMO

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. Deletion of fliD leads to non-motile mutant cells that are unable to assemble flagellar filaments and pentagon-shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella-deficient mutants (i.e., in the hook, rod, or MS-ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.


Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , Flagelos/química , Flagelina/química , Periplasma/química , Serina Endopeptidases/química , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Flagelos/genética , Mutação , Polímeros/química , Dobramento de Proteína , Serina Endopeptidases/genética
7.
FASEB J ; 33(1): 1033-1041, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30096040

RESUMO

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. Here we show that acetylated COX-2 also retains COX activity, forming predominantly 15 R-configuration PGs (70 or 62% 15 R, respectively, determined using radiolabeled substrate or LC-MS analysis). Although the Km of arachidonic acid for acetylated COX-2 was ∼3-fold lower than for uninhibited COX-2, the catalytic efficiency for PG formation by the acetylated enzyme was reduced 10-fold due to a concomitant decrease in Vmax. Aspirin increased 15 R-PGD2 but not 15 R-PGE2 in isolated human leukocytes activated with LPS to induce COX-2. 15 R-PGD2 inhibited human platelet aggregation induced by the thromboxane receptor agonist U46,619, and this effect was abrogated by an antagonist of the DP1 prostanoid receptor. We conclude that acetylation of Ser530 in COX-2 not only triggers formation of 15 R-HETE but also allows oxygenation and cyclization of arachidonic acid to a 15 R-PG endoperoxide. 15 R-PGs are novel products of aspirin therapy via acetylation of COX-2 and may contribute to its antiplatelet and other pharmacologic effects.-Giménez-Bastida, J. A., Boeglin, W. E., Boutaud, O., Malkowski, M. G., Schneider, C. Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.


Assuntos
Aspirina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Ácidos Hidroxieicosatetraenoicos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Acetilação , Células Cultivadas , Cromatografia Líquida , Humanos , Cinética , Leucócitos/metabolismo , Espectrometria de Massas
8.
Angew Chem Int Ed Engl ; 59(8): 3307-3314, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31854058

RESUMO

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. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX-2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen-dependent changes in COX-2 activity that are independent of protein expression within live macrophage cells.


Assuntos
Técnicas Biossensoriais/métodos , Ciclo-Oxigenase 2/química , Humanos , Simulação de Dinâmica Molecular
9.
Biochemistry ; 58(38): 3990-4002, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31469551

RESUMO

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. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial noncovalent complex, in the case of aspirin, and the "high-affinity" binding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial noncovalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15R-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co3+-protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-2). V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.


Assuntos
Aspirina/farmacologia , Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Animais , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Arginina/genética , Arginina/metabolismo , Cristalografia por Raios X , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/isolamento & purificação , Ensaios Enzimáticos , Histidina , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucina/genética , Leucina/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , Estereoisomerismo , Especificidade por Substrato , Fatores de Tempo
10.
Infect Immun ; 86(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29203544

RESUMO

A vaccine against Moraxella catarrhalis 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. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/imunologia , Moraxella catarrhalis/enzimologia , Infecções por Moraxellaceae/microbiologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Mapeamento de Epitopos , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Moraxella catarrhalis/química , Moraxella catarrhalis/genética , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/imunologia , Otite Média/imunologia , Otite Média/microbiologia
11.
J Biol Chem ; 291(29): 15069-81, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27226593

RESUMO

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. The underlying mechanism responsible for substrate-selective inhibition has remained elusive. We utilized structural and biophysical methods to evaluate flufenamic acid, meclofenamic acid, mefenamic acid, and tolfenamic acid for their ability to act as substrate-selective inhibitors. Crystal structures of each drug in complex with human COX-2 revealed that the inhibitor binds within the cyclooxygenase channel in an inverted orientation, with the carboxylate group interacting with Tyr-385 and Ser-530 at the top of the channel. Tryptophan fluorescence quenching, continuous-wave electron spin resonance, and UV-visible spectroscopy demonstrate that flufenamic acid, mefenamic acid, and tolfenamic acid are substrate-selective inhibitors that bind rapidly to COX-2, quench tyrosyl radicals, and reduce higher oxidation states of the heme moiety. Substrate-selective inhibition was attenuated by the addition of the lipid peroxide 15-hydroperoxyeicosatertaenoic acid. Collectively, these studies implicate peroxide tone as an important mechanistic component of substrate-selective inhibition by flufenamic acid, mefenamic acid, and tolfenamic acid.


Assuntos
Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Fenamatos/farmacologia , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Endocanabinoides/metabolismo , Fenamatos/química , Heme/química , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagênese Sítio-Dirigida , Peróxidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triptofano/química
12.
J Biol Chem ; 291(49): 25641-25655, 2016 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-27756840

RESUMO

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH2 PGHS-2 is a conformational heterodimer composed of allosteric (Eallo) and catalytic (Ecat) subunits. Fatty acids (FAs) bind to Arg-120 of Eallo increasing to different degrees, depending on the FA, the Vmax of its Ecat partner. We report here that movement of helical residues 120-122 and loop residues 123-129 of Eallo underlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen. An S121P substitution in both PGHS-2 monomers yields a variant (S121P/S121P PGHS-2) that has 1.7-1.8 times the Vmax of native PGHS-2 and is relatively insensitive to activation by FAs or inhibition by allosteric inhibitors. The S121P substitution in Eallo is primarily responsible for these effects. In X-ray crystal structures, the Cα atoms of helical residues 119-122 of S121P/S121P PGHS-2 are displaced from their normal positions. Additionally, the S121P/S121P PGHS-2 variants in which Pro-127 and Ser-541 are replaced by cysteines spontaneously forms Cys-127 to Cys-541 cross-links between monomers. This is unlike the corresponding native PGHS-2 variant and suggests that S121P substitutions also unhinge the loop involving residues 123-129. We conclude the following: (a) the region involving residues 120-129 of unoccupied Eallo tonically inhibits Ecat; (b) binding of an activating FA (e.g. arachidonic, palmitic, or oleic acid) to Eallo or an S121P substitution in Eallo repositions this region to increase Ecat activity; and (c) allosteric COX inhibitors act by preventing FA binding to Eallo and additionally by relocating Eallo residues to inhibit Ecat.


Assuntos
Inibidores de Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/química , Ácidos Graxos/química , Flurbiprofeno/química , Mutação de Sentido Incorreto , Naproxeno/química , Regulação Alostérica , Substituição de Aminoácidos , Domínio Catalítico , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Humanos , Estrutura Secundária de Proteína
13.
Biochemistry ; 55(8): 1226-38, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26859324

RESUMO

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). Ser-530 has also been shown to influence the stereochemistry for the addition of oxygen to 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 1.9, 2.0, and 2.4 Å, 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. On the basis of 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.


Assuntos
Acetilação/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/química , Aspirina/química , Ciclo-Oxigenase 2/química , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Cristalografia por Raios X , Ciclo-Oxigenase 2/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica
14.
J Biol Chem ; 290(50): 29820-33, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26515067

RESUMO

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). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes.


Assuntos
Oxigenases de Função Mista/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
15.
J Biol Chem ; 290(19): 12313-31, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25805501

RESUMO

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 ß-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. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the ß-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Porinas/química , Treponema pallidum/química , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/metabolismo , Temperatura Alta , Lipossomos/química , Microscopia Eletrônica , Microscopia de Fluorescência , Nanopartículas/química , Octoxinol , Peptídeos/química , Periplasma/metabolismo , Polietilenoglicóis/química , Desnaturação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Espalhamento de Radiação , Sífilis/microbiologia , Temperatura
16.
Infect Immun ; 84(2): 432-8, 2016 02.
Artigo em Inglês | MEDLINE | ID: mdl-26597985

RESUMO

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. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface of M. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement of M. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth of M. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Arginina/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Moraxella catarrhalis/crescimento & desenvolvimento , Moraxella catarrhalis/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Humanos , Moraxella catarrhalis/genética , Mutação , Proteínas Recombinantes/metabolismo
17.
Biochemistry ; 54(50): 7309-12, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26636181

RESUMO

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 distances obtained with DQC confirm that Tyr-385 and Tyr-504 radicals were generated in each monomer and accurately match the distances measured in COX-2 crystal structures.


Assuntos
Ciclo-Oxigenase 2/química , Análise Espectral/métodos , Tirosina/química , Dimerização , Modelos Moleculares
18.
J Struct Biol ; 189(1): 62-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25463020

RESUMO

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. While an X-ray crystal structure of IBP bound to COX-1 has been solved, no such structure exists for the cognate isoform COX-2. We have determined the crystal structure of muCOX-2 with a racemic mixture of (R/S)-IBP. Our structure reveals that only the S-isomer of IBP was bound, indicating that the S-isomer possesses higher affinity for COX-2 than the R-isomer. Mutational analysis of Arg-120 and Tyr-355 at the entrance of the cyclooxygenase channel confirmed their role in binding and inhibition of COX-2 by IBP. Our results provide the first atomic level detail of the interaction between IBP and COX-2.


Assuntos
Ciclo-Oxigenase 2/química , Ibuprofeno/química , Modelos Moleculares , Animais , Cristalização , Camundongos , Conformação Proteica
19.
J Struct Biol ; 192(1): 48-58, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26321039

RESUMO

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. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats.


Assuntos
Borrelia burgdorferi/enzimologia , Proteínas Periplásmicas/química , Proteínas Quinases/química , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Histidina Quinase , Modelos Moleculares , Dados de Sequência Molecular , Periplasma/enzimologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
20.
Infect Immun ; 82(11): 4758-66, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25156736

RESUMO

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. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Moraxella catarrhalis/enzimologia , Infecções por Moraxellaceae/microbiologia , Infecções Respiratórias/microbiologia , Animais , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Moraxella catarrhalis/genética , Moraxella catarrhalis/metabolismo , Família Multigênica , Mutação , Proteínas Recombinantes
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