RESUMEN
Bacteria have developed diverse strategies for defending their cell envelopes from external threats. In Firmicutes, one widespread strategy is to use Bce modules-membrane protein complexes that unite a peptide-detoxifying ABC transporter with a stress response coordinating two-component system. These modules provide specific, front-line defense for a wide variety of antimicrobial peptides and small molecule antibiotics as well as coordinate responses for heat, acid, and oxidative stress. Because of these abilities, Bce modules play important roles in virulence and the development of antibiotic resistance in a variety of pathogens, including Staphylococcus, Streptococcus, and Enterococcus species. Despite their importance, Bce modules are still poorly understood, with scattered functional data in only a small number of species. In this review, we will discuss Bce module structure in light of recent cryo-electron microscopy structures of the B. subtilis BceABRS module and explore the common threads and variations-on-a-theme in Bce module mechanisms across species. We also highlight the many remaining questions about Bce module function. Understanding these multifunctional membrane complexes will enhance our understanding of bacterial stress sensing and may point toward new therapeutic targets for highly resistant pathogens.
Asunto(s)
Proteínas Bacterianas , Pared Celular , Farmacorresistencia Bacteriana , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Pared Celular/metabolismo , Pared Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Antibacterianos/farmacología , Estrés Fisiológico , Regulación Bacteriana de la Expresión Génica , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Bacillus subtilis/metabolismoRESUMEN
Continual synthesis and remodeling of the peptidoglycan layer surrounding Gram-positive cells is essential for their survival. Diverse antimicrobial peptides target the lipid intermediates involved in this process. To sense and counteract assault from antimicrobial peptides, low G + C content gram-positive bacteria (Firmicutes) have evolved membrane protein complexes known as Bce-modules. These complexes consist minimally of an ABC transporter and a two-component system that work in tandem to perceive and confer resistance against antimicrobial peptides. In this mini-review I highlight recent breakthroughs in comprehending the structure and function of these unusual membrane protein complexes, with a particular focus on the BceAB-RS system present in Bacillus subtilis.
Asunto(s)
Antibacterianos , Péptidos Antimicrobianos , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Farmacorresistencia Bacteriana , Péptidos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , PercepciónRESUMEN
Gram-positive bacteria synthesize and secrete antimicrobial peptides that target the essential process of peptidoglycan synthesis. These antimicrobial peptides not only regulate the dynamics of microbial communities but are also of clinical importance as exemplified by peptides such as bacitracin, vancomycin, and daptomycin. Many gram-positive species have evolved specialized antimicrobial peptide sensing and resistance machinery known as Bce modules. These modules are membrane protein complexes formed by an unusual Bce-type ABC transporter interacting with a two-component system sensor histidine kinase. In this work, we provide the first structural insight into how the membrane protein components of these modules assemble into a functional complex. A cryo-EM structure of an entire Bce module revealed an unexpected mechanism of complex assembly, and extensive structural flexibility in the sensor histidine kinase. Structures of the complex in the presence of a non-hydrolysable ATP analog reveal how nucleotide binding primes the complex for subsequent activation. Accompanying biochemical data demonstrate how the individual membrane protein components of the complex exert functional control over one another to create a tightly regulated enzymatic system.
Asunto(s)
Péptidos Antimicrobianos , Péptidos , Histidina Quinasa , Transportadoras de Casetes de Unión a ATP , Proteínas de la MembranaRESUMEN
Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating ß-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via ß-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound ß-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.
Asunto(s)
Claudinas , Enterotoxinas , Animales , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Claudinas/metabolismo , Clostridium perfringens , Enterotoxinas/metabolismo , Epítopos/metabolismo , Humanos , Unión ProteicaRESUMEN
SignificanceMany gram-positive organisms have evolved an elegant solution to sense and resist antimicrobial peptides that inhibit cell-wall synthesis. These organisms express an unusual "Bce-type" adenosine triphosphate-binding cassette (ABC) transporter that recognizes complexes formed between antimicrobial peptides and lipids involved in cell-wall biosynthesis. In this work, we provide the first structural snapshots of a Bce-type ABC transporter trapped in different conformational states. Our structures and associated biochemical data provide key insights into the novel target protection mechanism that these unusual ABC transporters use to sense and resist antimicrobial peptides. The studies described herein set the stage to begin developing a comprehensive molecular understanding of the diverse interactions between antimicrobial peptides and conserved resistance machinery found across most gram-positive organisms.
Asunto(s)
Bacitracina , Farmacorresistencia Bacteriana , Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/metabolismo , Bacitracina/metabolismo , Bacitracina/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/ultraestructura , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructuraRESUMEN
ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Antineoplásicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/ultraestructura , Antineoplásicos/química , Transporte Biológico , Disulfuros/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/metabolismo , Ligandos , Mitoxantrona/química , Mitoxantrona/metabolismo , Modelos Biológicos , Estructura Secundaria de ProteínaRESUMEN
Gram-negative bacteria possess a dual-membrane envelope, which provides defense against environmental assault, as well as formidable resistance against antibiotics. Lipopolysaccharide (LPS) is the primary lipid component in the outermost membrane leaflet of most Gram-negative bacteria, and plays critical roles in cell envelope formation. Newly synthesized LPS at the cytoplasmic side of the inner membrane is flipped across the inner membrane and pushed across the periplasm by two ATP-binding cassette transporters, MsbA and LptB2FGC, respectively. Both transporters represent promising targets for developing new classes of antibiotics. In this review, we discuss recent advances in understanding the mechanism of LPS translocation driven by MsbA and LptB2FGC, with a particular focus on new findings from structural studies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Lipopolisacáridos/química , Modelos Moleculares , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Proteínas Portadoras , Lipopolisacáridos/metabolismo , Estructura Molecular , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.
Asunto(s)
Microscopía por Crioelectrón , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/ultraestructura , Pez Cebra , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cationes Monovalentes/metabolismo , Cloruros/metabolismo , Citosol/metabolismo , Síndrome de Gitelman/genética , Humanos , Transporte Iónico , Modelos Moleculares , Simulación de Dinámica Molecular , Potasio/metabolismo , Dominios Proteicos , Sodio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/química , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Pez Cebra/genéticaRESUMEN
Iron storage proteins are essential for cellular iron homeostasis and redox balance. Ferritin proteins are the major storage units for bioavailable forms of iron. Some organisms lack ferritins, and it is not known how they store iron. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo and show interactions underlying cargo-shell co-assembly. This compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. Our results reveal a new approach for survival in diverse habitats with limited or fluctuating iron availability via an iron storage system able to store 10 to 20 times more iron than ferritin.
Asunto(s)
Bacillaceae/metabolismo , Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Orgánulos/metabolismo , Bacillaceae/ultraestructura , Proteínas Bacterianas/química , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Ferritinas/química , Ferritinas/metabolismo , Homeostasis , Modelos Moleculares , Orgánulos/ultraestructura , Conformación ProteicaRESUMEN
In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide from the inner to the outer membrane. The ATP-binding cassette transporter LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane. The mechanism of the LptB2FG-LptC complex (LptB2FGC) and the role of LptC in lipopolysaccharide transport are poorly understood. Here we characterize the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ATP-binding cassette transporters. Our results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Unión Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Vanadatos/química , Vanadatos/metabolismo , Vanadatos/farmacologíaRESUMEN
Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Fusarium/química , Metarhizium/química , Animales , Caenorhabditis elegans/química , Calcio/metabolismo , Canales de Calcio/metabolismo , Cristalografía por Rayos X , Activación del Canal Iónico , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Reproducibilidad de los Resultados , SolubilidadRESUMEN
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. Vioxx is unique in that the inhibitor contains a methyl sulfone moiety in place of the sulfonamide moiety found in other coxibs such as celecoxib and valdecoxib. Here, new crystallization conditions were identified that allowed the structural determination of human COX-2 in complex with Vioxx and the structure was subsequently determined to 2.7â Å resolution. The crystal structure provides the first atomic level details of the binding of Vioxx to COX-2. As anticipated, Vioxx binds with its methyl sulfone moiety located in the side pocket of the cyclooxygenase channel, providing support for the isoform selectivity of this drug.
Asunto(s)
Celecoxib/química , Inhibidores de la Ciclooxigenasa 2/química , Ciclooxigenasa 2/química , Lactonas/química , Sulfonas/química , Secuencias de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retirada de Medicamento por Seguridad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/química , Ciclooxigenasa 2/química , Ácidos Grasos/química , Flurbiprofeno/química , Mutación Missense , Naproxeno/química , Regulación Alostérica , Sustitución de Aminoácidos , Dominio Catalítico , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Humanos , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Fenamatos/farmacología , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ciclooxigenasa 2/química , Ciclooxigenasa 2/genética , Endocannabinoides/metabolismo , Fenamatos/química , Hemo/química , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Peróxidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Triptófano/químicaRESUMEN
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.
Asunto(s)
Acetilación/efectos de los fármacos , Antiinflamatorios no Esteroideos/química , Aspirina/química , Ciclooxigenasa 2/química , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Cristalografía por Rayos X , Ciclooxigenasa 2/metabolismo , Humanos , Ratones , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
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.
Asunto(s)
Ciclooxigenasa 2/química , Análisis Espectral/métodos , Tirosina/química , Dimerización , Modelos MolecularesRESUMEN
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.
Asunto(s)
Ciclooxigenasa 2/química , Ibuprofeno/química , Modelos Moleculares , Animales , Cristalización , Ratones , Conformación ProteicaRESUMEN
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. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.
Asunto(s)
Biocatálisis , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Membrana Dobles de Lípidos/metabolismo , Nanotecnología/métodos , Animales , Ciclooxigenasa 2/química , Activación Enzimática/efectos de los fármacos , Humanos , Membrana Dobles de Lípidos/química , Ratones , Modelos Moleculares , Ácido Palmítico/farmacología , Fosfolípidos/metabolismo , Conformación ProteicaRESUMEN
The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H(2) from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co(3+)-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.