RESUMEN
The present work investigates the contribution of various second messenger systems to Ca(2+)-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca(2+) dependence of PS exposure was confirmed using the Ca(2+) ionophore bromo-A23187 to clamp intracellular Ca(2+) over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca(2+) loading in the presence of an outwardly directed electrochemical gradient for K(+) stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca(2+)] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca(2+)-induced PS exposure in RBCs, incubated in either HK or LK saline.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Calcio/farmacología , Eritrocitos/metabolismo , Fosfatidilserinas/metabolismo , Sistemas de Mensajero Secundario , Caribdotoxina/farmacología , Clotrimazol/farmacología , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Humanos , Bloqueadores de los Canales de Potasio/farmacología , Cloruro de Potasio/farmacologíaRESUMEN
Aromatic aldehydes like o-vanillin were designed to reduce the complications of sickle cell disease (SCD) by interaction with HbS, to reduce polymerisation and RBC sickling. Present results show that o-vanillin also directly affects RBC membrane permeability. Both the K(+)-Cl(-) cotransporter (KCC) and the Ca(2+)-activated K(+) channel (or Gardos channel) were inhibited with IC50 of about 0.3 and 1 mM, respectively, with activities almost completely abolished by 5 mM. Similar effects were observed in RBCs treated with the thiol reacting reagent N-ethylmaleimide or with the Ca(2+) ionophore A23187, to circumvent any action via HbS polymerisation. The deoxygenation-induced cation conductance (sometimes termed P(sickle)) was partially inhibited, whilst deoxygenation-induced exposure of phosphatidylserine was completely abrogated. Na(+)/K(+) pump activity was also reduced. Notwithstanding, o-vanillin stimulated K(+) efflux through an unidentified pathway and resulted in reduction in cell volume (as measured by wet weight-dry weight). These actions are relevant to understanding how aromatic aldehydes may affect RBC membrane permeability per se as well as HbS polymerisation and thereby inform design of compounds most efficacious in ameliorating the complications of SCD.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Benzaldehídos/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Potasio/metabolismo , Anemia de Células Falciformes/genética , Transporte Biológico/efectos de los fármacos , Calcimicina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Eritrocitos/patología , Hemoglobina Falciforme/genética , Homocigoto , Humanos , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Simportadores/metabolismo , Cotransportadores de K ClRESUMEN
Abstract Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.
Asunto(s)
Anemia de Células Falciformes/diagnóstico , Hemólisis/efectos de los fármacos , Aldehídos/farmacología , Membrana Celular/metabolismo , Citocalasina B/farmacología , Eritrocitos Anormales/efectos de los fármacos , Pruebas Hematológicas/métodos , Hemoglobinas/química , Hemoglobinas/genética , Hemólisis/genética , Humanos , Concentración de Iones de Hidrógeno , Polimerizacion , Pronóstico , Sacarosa/farmacología , Temperatura , Urea/farmacologíaRESUMEN
Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca²âº to low micromolar levels. The Ca²âº entry step has not been defined but a role for the deoxygenation-induced pathway, Psickle, is postulated. Partial Psickle inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), 4,4'-dithiocyano-2,2'-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn²âº and Gd³âº stimulated PS exposure to high levels. In the case of Zn²âº, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca²âº. The effect was completely abolished when Zn²âº (100 µM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn²âº.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Calcio/metabolismo , Eritrocitos Anormales/metabolismo , Oxígeno/farmacología , Fosfatidilserinas/metabolismo , Anemia de Células Falciformes/sangre , Eritrocitos Anormales/efectos de los fármacos , Gadolinio/farmacología , Hemoglobina Falciforme/metabolismo , Humanos , Zinc/farmacologíaRESUMEN
A mutant allele of the beta-chemokine receptor gene CCR5 bearing a 32-basepair (bp) deletion (denoted delta ccr5) which prevents cell invasion by the primary transmitting strain of HIV-1 has recently been characterized. Homozygotes for the mutation are resistant to infection, even after repeated high-risk exposures, but this resistance appears not to be total, as isolated cases of HIV-positive deletion homozygotes are now emerging. The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals. A gene frequency of approximately 10% was found for delta ccr5 in populations of European descent, but no mutant alleles were reported in indigenous non-European populations. As the total number of non-European samples surveyed was small in comparison with the Europeans the global distribution of this mutation is far from clear. We have devised a rapid PCR assay for delta ccr5 and used it to screen 3,342 individuals from a globally-distributed range of populations. We find that delta ccr5 is not confined to people of European descent but is found at frequencies of 2-5% throughout Europe, the Middle East and the Indian subcontinent (Fig. 1). Isolated occurrences are seen elsewhere throughout the world, but these most likely represent recent European gene flow into the indigenous populations. The inter-population differences in delta ccr5 frequency may influence the pattern of HIV transmission and so will need to be incorporated into future predictions of HIV levels.
Asunto(s)
Genética de Población , Receptores de Citocinas/genética , Receptores del VIH/genética , Eliminación de Secuencia , África/epidemiología , Animales , Asia/epidemiología , Europa (Continente)/epidemiología , Frecuencia de los Genes , Heterocigoto , Humanos , Judíos , Medio Oriente/epidemiología , Islas del Pacífico/epidemiología , Pan troglodytes/genética , Reacción en Cadena de la Polimerasa , Receptores CCR5 , Población Blanca/genéticaRESUMEN
The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved.
Asunto(s)
Anemia de Células Falciformes/sangre , Permeabilidad de la Membrana Celular , Membrana Eritrocítica/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Benzaldehídos/farmacología , Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Membrana Eritrocítica/efectos de los fármacos , Hemoglobina Falciforme/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Transporte Iónico , Magnesio/metabolismo , Manganeso/metabolismo , Potenciales de la Membrana , Moduladores del Transporte de Membrana/farmacología , Oxígeno/metabolismo , Técnicas de Placa-Clamp , Péptidos/farmacología , Potasio/metabolismo , Multimerización de Proteína , Rubidio/metabolismo , Sodio/metabolismo , Venenos de Araña/farmacología , Factores de TiempoRESUMEN
Acute neurovisceral attacks of porphyria can be life threatening. They are rare and notoriously difficult to diagnose clinically, but should be considered, particularly in female patients with unexplained abdominal pain, and associated neurological or psychiatric features or hyponatraemia. The diagnosis might be suggested by altered urine colour and can be confirmed by finding an elevated porphobilinogen concentration in fresh urine protected from light. Severe attacks require treatment with intravenous haem arginate and supportive management with safe drugs, including adequate analgesia. Intravenous glucose in water solutions are contraindicated as they aggravate hyponatraemia, which can prove fatal.
Asunto(s)
Dolor Abdominal/etiología , Alucinaciones/etiología , Hiponatremia/etiología , Porfiria Intermitente Aguda , Adolescente , Analgésicos/uso terapéutico , Arginina/uso terapéutico , Manejo de la Enfermedad , Resultado Fatal , Femenino , Hemo/uso terapéutico , Humanos , Monitoreo Fisiológico , Porfobilinógeno/orina , Porfiria Intermitente Aguda/complicaciones , Porfiria Intermitente Aguda/metabolismo , Porfiria Intermitente Aguda/fisiopatología , Porfiria Intermitente Aguda/terapia , Equilibrio HidroelectrolíticoRESUMEN
Sickle cell disease (SCD) presents a significant global health problem. At present there is no effective treatment, with most being supportive for its associated complications such as the vaso-occlusive crises that result from increased cell adhesion. Hypoxic sickle cells have previously shown greater phosphatidylserine (PS) exposure and oxidative damage, as well as being notably "stickier" suggesting that increased cell cohesion and adhesion to the blood vessel endothelium is a possible mechanism for vaso-occlusion. The present work uses the hybrid technique of atomic force microscopy nano-infrared spectroscopy (AFM-IR) to probe changes to the coefficient of friction and C-O IR intensity in SCD on a nanoscale for dried red blood cells (RBCs) fixed under conditions of hypoxia and correlates these observations with adhesive interactions at the membrane. Using functionalised AFM tips, it has been possible to probe adhesive interactions between hydrophilic and hydrophobic moieties exposed at the surface of the dried RBCs fixed under different oxygenation states and for different cell genotypes. The results are consistent with greater PS-exposure and oxidative damage in hypoxic sickle cells, as previously proposed, and also show strong correlation between localised oxidative damage and increased adhesion. A mechanistic explanation involving significant lipid tail disruption as a result of oxidative action, in combination with differing concentrations of externalised PS lipids, is proposed to explain the observed adhesion behaviour of each type of cell.
Asunto(s)
Anemia de Células Falciformes , Adhesión Celular , Eritrocitos , Humanos , Microscopía de Fuerza Atómica , Análisis EspectralRESUMEN
Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Permeabilidad de la Membrana Celular , Eritrocitos/patología , Hemoglobina C/genética , Enfermedad de la Hemoglobina SC/metabolismo , Hemoglobina Falciforme/genética , Anemia de Células Falciformes/genética , Calcio/metabolismo , Cationes/metabolismo , Niño , Fenómenos Electrofisiológicos , Eritrocitos/metabolismo , Hemoglobina C/metabolismo , Enfermedad de la Hemoglobina SC/genética , Hemoglobina Falciforme/metabolismo , Hemólisis , Heterocigoto , Humanos , Técnicas de Placa-ClampRESUMEN
Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.
Asunto(s)
Anemia de Células Falciformes/sangre , Eritrocitos/metabolismo , Fosfatidilserinas/sangre , Benzofenantridinas/farmacología , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Membrana Eritrocítica/química , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/sangre , Pirazinas/administración & dosificación , Pirazinas/farmacología , Venenos de Araña/farmacología , Tiadiazoles/administración & dosificación , Tiadiazoles/farmacologíaRESUMEN
Fractal surfaces can be used to characterize the roughness or irregularity of protein surfaces. The degree of irregularity of a surface may be described by the fractal dimension D. For protein surfaces defined with probes in the range of 1.0 to 3.5 angstroms in radius, D is approximately 2.4 or intermediate between the value for a completely smooth surface (D = 2) and that for a completely space-filling surface (D = 3). Individual regions of proteins show considerable variation in D. These variations may be related to structural features such as active sites and subunit interfaces, suggesting that surface texture may be a factor influencing molecular interactions.
Asunto(s)
Proteínas/metabolismo , Carboxipeptidasas/metabolismo , Carboxipeptidasas A , Muramidasa/metabolismo , Ribonucleasa Pancreática/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Structural models for the nitrogenase FeMo-cofactor and P-clusters are proposed based on crystallographic analysis of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii at 2.7 angstrom resolution. Each center consists of two bridged clusters; the FeMo-cofactor has 4Fe:3S and 1Mo:3Fe:3S clusters bridged by three non-protein ligands, and the P-clusters contain two 4Fe:4S clusters bridged by two cysteine thiol ligands. Six of the seven Fe sites in the FeMo-cofactor appear to have trigonal coordination geometry, including one ligand provided by a bridging group. The remaining Fe site has tetrahedral geometry and is liganded to the side chain of Cys alpha 275. The Mo site exhibits approximate octahedral coordination geometry and is liganded by three sulfurs in the cofactor, two oxygens from homocitrate, and the imidazole side chain of His alpha 442. The P-clusters are liganded by six cysteine thiol groups, two which bridge the two clusters, alpha 88 and beta 95, and four which singly coordinate the remaining Fe sites, alpha 62, alpha 154, beta 70, and beta 153. The side chain of Ser beta 188 may also coordinate one iron. The polypeptide folds of the homologous alpha and beta subunits surrounding the P-clusters are approximately related by a twofold rotation that may be utilized in the binding interactions between the MoFe-protein and the nitrogenase Fe-protein. Neither the FeMo-cofactor nor the P-clusters are exposed to the surface, suggesting that substrate entry, electron transfer, and product release must involve a carefully regulated sequence of interactions between the MoFe-protein and Fe-protein of nitrogenase.
Asunto(s)
Azotobacter vinelandii/enzimología , Modelos Moleculares , Molibdoferredoxina/química , Nitrogenasa/química , Sitios de Unión , Fenómenos Químicos , Química Física , Cristalización , Transporte de Electrón , Proteínas Hierro-Azufre/química , Sustancias Macromoleculares , Estructura Molecular , Nitrógeno/metabolismo , Oxidación-Reducción , Análisis Espectral , Difracción de Rayos XRESUMEN
Membrane-exposed residues are more hydrophobic than buried interior residues in the transmembrane regions of the photosynthetic reaction center from Rhodobacter sphaeroides. This hydrophobic organization is opposite to that of water-soluble proteins. The relative polarities of interior and surface residues of membrane and water soluble proteins are not simply reversed, however. The hydrophobicities of interior residues of both membrane and water-soluble proteins are comparable, whereas the bilayer-exposed residues of membrane proteins are more hydrophobic than the interior residues, and the aqueous-exposed residues of water-soluble proteins are more hydrophilic than the interior residues. A method of sequence analysis is described, based on the periodicity of residue replacement in homologous sequences, that extends conclusions derived from the known atomic structure of the reaction center to the more extensive database of putative transmembrane helical sequences.
Asunto(s)
Proteínas Bacterianas , Proteínas de la Membrana , Rhodobacter sphaeroides/ultraestructura , Membrana Celular/análisis , Fenómenos Químicos , Química Física , Análisis de Fourier , Proteínas del Complejo del Centro de Reacción Fotosintética , Conformación Proteica , Solubilidad , AguaRESUMEN
Structures recently proposed for the FeMo-cofactor and P-cluster pair of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii have been crystallographically verified at 2.2 angstrom resolution. Significantly, no hexacoordinate sulfur atoms are observed in either type of metal center. Consequently, the six bridged iron atoms in the FeMo-cofactor are trigonally coordinated by nonprotein ligands, although there may be some iron-iron bonding interactions that could provide a fourth coordination interaction for these sites. Two of the cluster sulfurs in the P-cluster pair are very close together (approximately 2.1 angstroms), indicating that they form a disulfide bond. These findings indicate that a cavity exists in the interior of the FeMo-cofactor that could be involved in substrate binding and suggest that redox reactions at the P-cluster pair may be linked to transitions of two cluster-bound sulfurs between disulfide and sulfide oxidation states.
Asunto(s)
Azotobacter vinelandii/enzimología , Hierro/química , Molibdoferredoxina/química , Nitrogenasa/química , Azufre/química , Modelos Moleculares , Oxidación-Reducción , Difracción de Rayos XRESUMEN
Structures of the protein-chromophore complex and the apoprotein form of neocarzinostatin were determined at 1.8 angstrom resolution. Neocarzinostatin is composed of a labile chromophore with DNA-cleaving activity and a stabilizing protein. The chromophore displays marked nonlinearity of the triple bonds and is bound noncovalently in a pocket formed by the two protein domains. The chromophore pi-face interacts with the phenyl ring edges of Phe52 and Phe78. The amino sugar and carbonate groups of the chromophore are solvent exposed, whereas the epoxide, acetylene groups, and carbon C-12, the site of nucleophilic thiol addition during chromophore activation, are unexposed. The position of the amino group of the chromophore carbohydrate relative to C-12 supports the idea that the amino group plays a role in thiol activation.
Asunto(s)
Cinostatina/química , Apoproteínas/química , Gráficos por Computador , Simulación por Computador , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.
Asunto(s)
Aldehído Oxidorreductasas/química , Archaea/enzimología , Coenzimas , Compuestos Organometálicos/química , Pterinas/química , Tungsteno/química , Aldehído Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Gráficos por Computador , Cristalografía por Rayos X , Estabilidad de Enzimas , Compuestos Ferrosos , Metaloproteínas/análisis , Metaloproteínas/química , Modelos Moleculares , Datos de Secuencia Molecular , Cofactores de Molibdeno , Compuestos Organometálicos/análisis , Oxidación-Reducción , Conformación Proteica , Estructura Secundaria de Proteína , Pteridinas/análisis , Pteridinas/química , Pterinas/análisis , Propiedades de Superficie , Temperatura , Tungsteno/análisisRESUMEN
Bleomycin hydrolase is a cysteine protease that hydrolyzes the anticancer drug bleomycin. The homolog in yeast, Gal6, has recently been identified and found to bind DNA and to act as a repressor in the Gal4 regulatory system. The crystal structure of Gal6 at 2.2 A resolution reveals a hexameric structure with a prominent central channel. The papain-like active sites are situated within the central channel, in a manner resembling the organization of active sites in the proteasome. The Gal6 channel is lined with 60 lysine residues from the six subunits, suggesting a role in DNA binding. The carboxyl-terminal arm of Gal6 extends into the active site cleft and may serve a regulatory function. Rather than each residing in distinct, separable domains, the protease and DNA-binding activities appear structurally intertwined in the hexamer, implying a coupling of these two activities.
Asunto(s)
Cisteína Endopeptidasas/química , ADN/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Gráficos por Computador , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The molybdoenzyme dimethylsulfoxide (DMSO) reductase contributes to the release of dimethylsulfide, a compound that has been implicated in cloud nucleation and global climate regulation. The crystal structure of DMSO reductase from Rhodobacter sphaeroides reveals a monooxo molybdenum cofactor containing two molybdopterin guanine dinucleotides that asymmetrically coordinate the molybdenum through their dithiolene groups. One of the pterins exhibits different coordination modes to the molybdenum between the oxidized and reduced states, whereas the side chain oxygen of Ser147 coordinates the metal in both states. The change in pterin coordination between the Mo(VI) and Mo(IV) forms suggests a mechanism for substrate binding and reduction by this enzyme. Sequence comparisons of DMSO reductase with a family of bacterial oxotransferases containing molybdopterin guanine dinucleotide indicate a similar polypeptide fold and active site with two molybdopterins within this family.
Asunto(s)
Coenzimas/química , Proteínas Hierro-Azufre , Metaloproteínas/química , Oxidorreductasas/química , Pteridinas/química , Rhodobacter sphaeroides/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Cofactores de Molibdeno , Oxidación-Reducción , Oxidorreductasas/metabolismo , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/química , Sitios de Unión , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cristalización , Cristalografía por Rayos X , Heparina/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Conformación ProteicaRESUMEN
The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc1.