RESUMEN
Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ â¼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
Asunto(s)
Descubrimiento de Drogas , Glutamato Deshidrogenasa/ultraestructura , Isocitrato Deshidrogenasa/ultraestructura , L-Lactato Deshidrogenasa/ultraestructura , Aminoquinolinas/química , Aminoquinolinas/farmacología , Animales , Bovinos , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/química , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Modelos Moleculares , Conformación Proteica , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The human muscle isoform of lactate dehydrogenase (hLDH5) is one of the key enzymes of the glycolytic process. It is overexpressed in metastatic cancer cells and is linked to the vitality of tumors in hypoxic conditions. With the aim of identifying new hLDH5 inhibitors, a fully automated docking-based virtual screening platform was developed by considering different protein conformations and the consensus docking strategy. In order to verify the reliability of the reported platform, a small database of about 10,000 compounds was filtered by using this method, and the top-ranked compounds were tested for their hLDH5 inhibition activity. Enzymatic assays revealed that, among the ten selected compounds, two proved to efficiently inhibit enzyme activity with IC50 values in the micromolar range. These results demonstrate the validity of the methodologies we followed, encouraging the application of larger virtual screening studies and further refinements of the platform. Furthermore, the two active compounds herein described may be considered as interesting leads for the development of new and more efficient LDH inhibitors.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/ultraestructura , Músculo Esquelético/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Glucólisis/fisiología , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Músculo Esquelético/enzimología , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica/fisiología , Conformación ProteicaRESUMEN
Nanomaterials in biological solutions are known to interact with proteins and have been documented to affect protein function, such as enzyme activity. Understanding the interactions of nanoparticles with biological components at the molecular level will allow for rational designs of nanomaterials for use in medical technologies. Here we present the first detailed molecular mechanics model of functionalized gold nanoparticle (NP) interacting with an enzyme (L-lactate dehydrogenase (LDH) enzyme). Molecular dynamics (MD) simulations of the response of LDH to the NP binding demonstrate that although atomic motions (dynamics) of the main chain exhibit only a minor response to the binding, the dynamics of side chains are significantly constrained in all four active sites that predict alteration in kinetic properties of the enzyme. It is also demonstrated that the 5 nm gold NPs cause a decrease in the maximal velocity of the enzyme reaction (V(max)) and a trend towards a reduced affinity (increased K(m)) for the ß-NAD binding site, while pyruvate enzyme kinetics (K(m) and V(max)) are not significantly altered in the presence of the gold NPs. These results demonstrate that modeling of NP:protein interactions can be used to understand alterations in protein function.
Asunto(s)
Oro/química , L-Lactato Deshidrogenasa/química , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Modelos Químicos , Simulación de Dinámica Molecular , Simulación por Computador , Activación Enzimática , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/ultraestructura , L-Lactato Deshidrogenasa/ultraestructura , Nanoconjugados/química , Nanoconjugados/ultraestructura , Tamaño de la Partícula , Unión ProteicaRESUMEN
BACKGROUND: Although commercial skin substitutes are widely available, its use remains challenging at surgery and postoperatively. The high cost is also prohibitive. We designed and characterized a scaffold for dermal replacement, using advanced nanocomposite materials, which are known to have unique nanoscale features that enhance cellular behavior. METHODS: A bilayered scaffold was developed using the nanocomposite, polyhedral oligomeric silsesquioxane, incorporated into poly(caprolactone-urea)urethane, resulting in a mechanically robust bioabsorbable polymer; forming the inner layer, which was designed with a range of porosities. The removable outer layer contained nanosilver. Tensile testing, surface tension, permeability, and scanning electron microscopy were performed. Optimal pore morphology for cellular proliferation was elucidated through adipose tissue-derived stem cell culture and a cell viability assay. All tests were repeated on Integra Dermal Regeneration Template. RESULTS: The physical construct was easy to handle and clinically applicable. Macroporosity and permeability of scaffolds was demonstrated, confirmed by scanning electron microscopy. Both tensile strength and surface tension were comparable with skin; outer layer demonstrated hydrophobicity and inner layer showed hydrophilicity. Cell assay confirmed cellular proliferation onto the scaffold, comparable with Integra. CONCLUSIONS: We demonstrate that a porous bilayered dermal scaffold could form the basis of a new generation of skin substitute that is both mechanically robust and harbors the ability for enhancing cell regeneration.
Asunto(s)
Compuestos de Organosilicio/química , Piel , Andamios del Tejido , Tejido Adiposo/ultraestructura , Fenómenos Biomecánicos , Supervivencia Celular , Humanos , L-Lactato Deshidrogenasa/ultraestructura , Microscopía Electrónica de Rastreo , Nanocompuestos/ultraestructura , Piel/ultraestructura , Piel Artificial , Células Madre/ultraestructuraRESUMEN
We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to "fold" to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.
Asunto(s)
L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/ultraestructura , Modelos Químicos , Modelos Moleculares , Mapeo de Interacción de Proteínas/métodos , Sitios de Unión , Simulación por Computador , Activación Enzimática , Ligandos , Unión ProteicaRESUMEN
In order to investigate the molecular mechanism of the F-actin conformation modifying activity [Biochem. Biophys. Res. Commun. 319 (2004) 78] of actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910], the ultrastructure and the regulatory mechanism of the activity were further examined. Interestingly, a novel oligomeric grapple-like structure of 10-12 subunits with an ATP-dependent opening was observed. ATP regulates the opening and closing of the "gate" that forms the opening within oligomeric Aip2p/Dld2p, where binding to the substrate occurs while in the open form. In the presence of ATP (open state of oligomeric Aip2p/Dld2p), oligomeric Aip2p/Dld2p bound the F-actin fiber within the opening, whereas in the absence of ATP (closed state of oligomeric Aip2p/Dld2p), no binding was observed. Simultaneously, the oligomeric Aip2p/Dld2p increased the trypsin susceptibility of F-actin in an ATP-dependent manner. Use of the non-hydrolyzable ATP analogue AMP-PNP yielded similar results to those observed with ATP, suggesting that ATP binding rather than ATP hydrolysis is required for the protein conformation modifying reaction of oligomeric Aip2p/Dld2p. Endogenous Aip2p/Dld2p purified from Saccharomyces cerevisiae also exhibited such protein conformation modifying activity, but monomeric Aip2p/Dld2p with a C-terminal coiled-coil region-truncation failed to exhibit the activity. These data suggest that the oligomerization of Aip2p/Dld2p, which exhibits the unique grapple-like structure with an ATP-dependent opening, is required for the F-actin conformation modifying activity.
Asunto(s)
Actinas/química , Adenosina Trifosfato/química , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Dimerización , L-Lactato Deshidrogenasa (Citocromo) , Polímeros/química , Unión Proteica , Conformación ProteicaRESUMEN
L-Lactate dehydrogenase (LDH) from Lactobacillus pentosus is a non-allosteric enzyme, which shows, however, high sequence similarity to allosteric LDHs from certain bacteria. To elucidate the structural basis of the absence of allostery of L. pentosus LDH (LPLDH), we determined the crystal structure of LPLDH at 2.3 A resolution. Bacterial LDHs are tetrameric enzymes composed of identical subunits and exhibit 222 symmetry. The quaternary structure of LPLDH was similar to the active conformation of allosteric LDHs. Structural analysis revealed that the subunit interfaces of LPLDH are optimized mainly through hydrophilic interactions rather than hydrophobic interactions, compared with other LDHs. The subunit interfaces of LPLDH are more specifically stabilized by increased numbers of intersubunit salt bridges and hydrogen bonds, and higher geometrical complementarity. Such high specificity at the subunit interfaces should hinder the rearrangement of the quaternary structure needed for allosteric regulation and thus explain the "non-allostery" of LPLDH.
Asunto(s)
L-Lactato Deshidrogenasa/química , Lactobacillus/enzimología , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/ultraestructura , L-Lactato Deshidrogenasa (Citocromo) , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de ProteínaRESUMEN
UNLABELLED: The purpose of this study is to evaluate the effect of C18 and C20 long chain fatty acids on tight junction permeability in a model of intestinal epithelium. METHODS: Confluent Caco-2 cells on porous filters with double chamber system were used to measure fluorescein sulfonic acid (FS) permeability and transepithelial electrical resistance (TEER). Lactate dehydrogenase release and ultrastructure were evaluated. Effect of 200 microM eicosapentaenoic acid (EPA, C20:5 n-3), arachidonic acid (AA, C20: 4 n-6), alpha-linoleic acid (ALA, C18: 3 n-3), linoleic acid (LA, C18: 2 n-6), or oleic acid (OA, C18: 1 n-9) enrichment in the culture medium during 24 hours were compared. The effect of the cyclooxygenase inhibitor, indomethacin, lipoxygenase inhibitors, NDGA or AA861, and antioxidant, BHT, was evaluated as a mechanism to change tight junction permeability. RESULTS: Caco-2 cells formed polarized columnar epithelial cells with densely packed microvilli and well developed junctional complexes. Addition of EPA enhanced FS permeability to 3.0+/-1.6-fold and lowered TEER to 0.59+/-1.2-fold vs. control with concentration dependency without cell injury (P<0.01-0.05). OA, AA or LA did not change, but ALA enhanced tight junction permeability. Indomethacin and AA861 normalized the changes mediated by EPA. CONCLUSIONS: EPA affects tight junction permeability in intestinal monolayer cells specifically and concentration dependently via cyclooxygenase and lipoxygenase products.
Asunto(s)
Eicosanoides/farmacología , Ácidos Grasos Insaturados/farmacología , Fluoresceínas/análisis , Uniones Estrechas/efectos de los fármacos , Células CACO-2 , Membrana Celular , Permeabilidad de la Membrana Celular , Impedancia Eléctrica , Ácidos Grasos/análisis , Humanos , Mucosa Intestinal , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/ultraestructura , Lipooxigenasa , Microscopía Electrónica , Permeabilidad , Prostaglandina-Endoperóxido SintasasRESUMEN
Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel-permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02-50 mg/ml) and temperature (20-80 degrees C). Rechromatography confirms this finding: the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species; increased temperature favors octamer formation. Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation causes irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion: image processing shows that the dumb-bell-shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.
Asunto(s)
Bacterias Anaerobias Gramnegativas/enzimología , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/aislamiento & purificación , Sitios de Unión , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli , Guanidina , Guanidinas , L-Lactato Deshidrogenasa/ultraestructura , Luz , Sustancias Macromoleculares , Microscopía Electrónica , Peso Molecular , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Dispersión de Radiación , EspectrofotometríaRESUMEN
The form of presentation of a new case of Melas Syndrome is described, together with a pathological and neuroimage study, including clinical development over a 3 year period. The usefulness of MR should be underlined here, given clinical doubts, and also normality in the EMG early phases of and the association with obstructive hypertrophic miocardiopathy.
Asunto(s)
Síndrome MELAS/diagnóstico , Imagen por Resonancia Magnética/métodos , Adolescente , Encéfalo/irrigación sanguínea , Encéfalo/fisiopatología , Cardiomiopatía Hipertrófica/complicaciones , Humanos , L-Lactato Deshidrogenasa/ultraestructura , Síndrome MELAS/complicaciones , Síndrome MELAS/fisiopatología , Masculino , Mitocondrias/ultraestructura , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.
Asunto(s)
L-Lactato Deshidrogenasa/química , Ribosomas/química , Escherichia coli , Técnicas In Vitro , L-Lactato Deshidrogenasa/ultraestructura , Conformación Proteica , Desnaturalización Proteica , ARN Ribosómico/química , Ribosomas/ultraestructuraRESUMEN
The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Nucleótidos/metabolismo , Conformación Proteica , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Chaperonina 10 , Chaperonina 60 , Geobacillus stearothermophilus , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/ultraestructura , Sustancias Macromoleculares , Proteínas Recombinantes , TermodinámicaRESUMEN
The three-dimensional structure of allosteric L-lactate dehydrogenase from Bifidobacterium longum, the first example of a T-state structure of L-lactate dehydrogenase, has been determined to 2.0 A. A comparative study of this structure with the previously reported R-state structure from Bacillus stearothermophilus has revealed the allosteric activation mechanism of the bacterial L-lactate dehydrogenase. The fructose 1,6-bisphosphate-induced conformational change at the effector site and the substrate affinity change at the activity site are clearly shown at a molecular level. Coupling of these changes can be simply explained by a set of concerted rotations between subunits in the tetramer of the enzyme. This T to R transition is the first example for a tetrameric allosteric protein where the rotations occur around each of three axes of symmetry.
Asunto(s)
Bifidobacterium/enzimología , L-Lactato Deshidrogenasa/metabolismo , Regulación Alostérica , Sitios de Unión , Cristalografía , Activación Enzimática , Fructosadifosfatos/farmacología , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/ultraestructura , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Sulfatos/químicaRESUMEN
We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).
Asunto(s)
Geobacillus stearothermophilus/enzimología , L-Lactato Deshidrogenasa/ultraestructura , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía , Análisis Mutacional de ADN , Fructosafosfatos/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Aceleradores de Partículas , Difracción de Rayos XAsunto(s)
Peces Killi/fisiología , Adaptación Biológica , Alelos , Animales , Gráficos por Computador , Ambiente , Variación Genética , Genética de Población , Geografía , Isoenzimas/genética , Cinética , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/ultraestructura , Modelos Moleculares , Filogenia , NataciónRESUMEN
By exhaustive structural comparisons, we have found that about one-third of the alpha-helix-turn-beta-strand polypeptides in alpha-beta barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the alpha-helix and the beta-strand is somewhat constrained, and second, the beta-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the alpha-helix. The geometry of the turn does not seem to be a major determinant of the alpha-beta unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the alpha-helix and the beta-strands are very long. It also occurs much less frequently in flat-sheet alpha-beta proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which alpha-beta barrels are constructed.