RESUMEN
This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule" strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a para-nitrophenyl phosphonate group that is a covalent inhibitor for cutinase. This enzyme is a serine hydrolase and reacts efficiently with the phosphonate to give a new ester linkage at the Ser-120 residue in the active site of the enzyme. Negative-stain transmission electron microscopy (TEM) images confirmed the architecture of the four-armed megamolecules. These cutinase tetramers were also characterized by X-ray crystallography, which confirmed the active-site serine-phosphonate linkage by electron-density maps. Molecular dynamics simulations of the tetracutinase megamolecules using three different force field setups were performed and compared with the TEM observations. Using the Amberff99SB-disp + pH7 force field, the two-dimensional projection distances of the megamolecules were found to agree with the measured dimensions from TEM. The study described here, which combines high-resolution characterization with molecular dynamics simulations, will lead to a comprehensive understanding of the molecular structures and dynamics for this new class of molecules.
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Organofosfonatos , Dominio Catalítico , Cristalografía por Rayos X , Estructura Molecular , Dominios ProteicosRESUMEN
This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.
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Microscopía por Crioelectrón , Investigación/organización & administración , Microscopía por Crioelectrón/instrumentación , Flujo de TrabajoRESUMEN
The "vanishing bone" syndromes represent a group of rare skeletal disorders characterized by osteolysis and joint destruction, which can mimic severe rheumatoid arthritis. Winchester syndrome was one of the first recognized autosomal-recessive, multicentric forms of the disorder. It was originally described nearly 50 years ago in two sisters with a severe crippling osteolysis. Using cultured fibroblasts from the proband, we have now identified homozygous mutations in membrane type-1 metalloproteinase (MT1-MMP or MMP14). We demonstrate that the resulting hydrophobic-region signal-peptide substitution (p.Thr17Arg) decreases MT1-MMP membrane localization with consequent impairment of pro-MMP2 activation, and we propose a structure-based mechanism for this effect.
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Anomalías Múltiples/genética , Artritis/genética , Contractura/genética , Opacidad de la Córnea/genética , Trastornos del Crecimiento/genética , Síndrome de Hajdu-Cheney/genética , Metaloproteinasa 14 de la Matriz/genética , Osteólisis/genética , Osteoporosis/genética , Anomalías Múltiples/diagnóstico por imagen , Secuencia de Aminoácidos , Contractura/diagnóstico por imagen , Opacidad de la Córnea/diagnóstico por imagen , Femenino , Trastornos del Crecimiento/diagnóstico por imagen , Humanos , Modelos Moleculares , Mutación , Osteólisis/diagnóstico por imagen , Osteoporosis/diagnóstico por imagen , RadiografíaRESUMEN
Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.
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Acetamidas/química , Acetamidas/farmacología , Antivirales/química , Antivirales/farmacología , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Acetamidas/síntesis química , Antivirales/síntesis química , Humanos , Modelos Moleculares , Síndrome Respiratorio Agudo Grave/virología , Relación Estructura-ActividadRESUMEN
Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
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Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Biocatálisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Mutagénesis Sitio-Dirigida , Adenosina Difosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas Quinasas Asociadas a Muerte Celular , Pruebas de Enzimas , Glutamina/genética , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Estructura Secundaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad , Valina/genéticaRESUMEN
Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.
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Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Evaluación Preclínica de Medicamentos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Secuencia de Aminoácidos , Proteasas 3C de Coronavirus , Cristalización , Cisteína Endopeptidasas/genética , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Especificidad por SustratoRESUMEN
Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is critical to potency. Inhibitor 10 with a 5-chloropyridinyl ester at position 4 of the indole ring is the most potent inhibitor with a SARS-CoV 3CLpro IC(50) value of 30 nM and an antiviral EC(50) value of 6.9 microM. Molecular docking studies have provided possible binding modes of these inhibitors.
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Antivirales/síntesis química , Química Farmacéutica/métodos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacología , Dominio Catalítico , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/química , Diseño de Fármacos , Ésteres , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Unión ProteicaRESUMEN
Infantile systemic hyalinosis (ISH) is a rare, progressive autosomal recessive disease, which is usually fatal by the age of 2 years. Clinical onset typically occurs within the first few weeks of life. The disease is characterized by joint contractures, osteopenia, failure to thrive, gingival hypertrophy, diarrhea, protein-losing enteropathy, and frequent infections. Dermatologic manifestations include thickened skin, hyperpigmentation, perianal nodules, and facial papules. Histopathology shows hyaline deposits in the dermis and visceral organs. We describe a patient with ISH confirmed by clinical and histopathologic findings, as well as DNA sequence analysis, which revealed a novel homozygous T118K mutation in the CMG2 gene.
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Contractura/patología , Artropatías/patología , Enfermedades Musculares/patología , Enfermedades de la Piel/patología , Sustitución de Aminoácidos , Contractura/genética , Diarrea/patología , Resultado Fatal , Femenino , Humanos , Lactante , Artropatías/genética , Proteínas de la Membrana/genética , Enfermedades Musculares/genética , Receptores de Péptidos , Enfermedades de la Piel/genéticaRESUMEN
Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 A resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.
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Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Diseño de Fármacos , Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/efectos de los fármacos , Sulfonamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Línea Celular , Cristalografía por Rayos X , Darunavir , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , VIH-1/enzimología , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.
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Calcio/metabolismo , Membrana Celular/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/enzimología , Dicroismo Circular , Glioma/enzimología , Metaloendopeptidasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Ratas , Especificidad por SustratoRESUMEN
The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150's exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.
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Encéfalo/efectos de los fármacos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/psicología , Animales , Aprendizaje por Asociación/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/fisiopatología , Ratones Transgénicos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/fisiología , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas Sprague-Dawley , Memoria Espacial/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure-activity relationships within S(1'), S(1), and S(2) enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.
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Acetamidas/química , Acetamidas/farmacología , Descubrimiento de Drogas , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Acetamidas/síntesis química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.
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Encéfalo/enzimología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/química , Piridazinas/farmacología , Piridinas/química , Piridinas/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dominio Catalítico , Línea Celular , Diseño de Fármacos , Humanos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Proteína Quinasa 14 Activada por Mitógenos/química , Modelos Moleculares , Fragmentos de Péptidos/toxicidad , Inhibidores de Proteínas Quinasas/síntesis química , Piridazinas/síntesis química , Piridinas/síntesis químicaRESUMEN
Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors are described. These inhibitors were designed and synthesized based upon our X-ray crystal structure of inhibitor 1 bound to SARS-CoV 3CLpro. Incorporation of Boc-Ser as the P(4)-ligand resulted in enhanced SARS-CoV 3CLpro inhibitory activity. Structural analysis of the inhibitor-bound X-ray structure revealed high binding affinity toward the enzyme.
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Imitación Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteasas 3C de Coronavirus , Cristalografía por Rayos X , Cisteína Endopeptidasas , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis químicaRESUMEN
The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were determined by X-ray crystallography in a resolution range between 2.3 and 2.7 A. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 A resolution. The native structure revealed a scissile bond angle (tau) of 158 degrees, which is close to the requisite 180 degrees "in-line" geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine), and G8(uridine) folded properly, but exhibited nonideal scissile bond geometries (tau ranging from 118 degrees to 93 degrees) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A-1 sugar pucker. In this regard, variants A8 and U8 appeared to represent nonproductive ground states in which their 2'-OH groups mimicked the pro-R, nonbridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A-1, lending support to the hypothesis that solvent may play an important role in the reaction.
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Fósforo/química , Fósforo/metabolismo , ARN Catalítico/química , ARN Catalítico/metabolismo , Agua/metabolismo , Sitios de Unión , Unión Competitiva , Cinética , Modelos Químicos , Conformación de Ácido Nucleico , ARN Catalítico/genética , Relación Estructura-ActividadRESUMEN
The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.
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Metales/química , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN/química , Uracilo/química , Cristalización , Modelos Moleculares , Estructura Molecular , ARN/genética , ARN Catalítico/genética , Difracción de Rayos XRESUMEN
The hairpin ribozyme is a naturally occurring catalytic RNA composed of two helix-loop-helix domains, A and B, that dock to form the biologically active enzyme. Previously, the crystal structure of the hairpin has been solved as a four-way helical junction that incorporated the U1A protein as an artificial crystal-packing motif [Rupert & Ferré-D'Amaré (2001), Nature (London), 410, 780-786]. Here, the crystallization of a minimal junctionless hairpin ribozyme 64-mer is reported in the absence of protein. Crystals grow in space group P6(1)22, with unit-cell parameters a = 93.1, c = 123.2 A. Complete diffraction data have been collected to 3.35 A resolution. Structural analysis should provide details of intermolecular RNA docking, including the ground-state conformations of the U(39)C mutation relevant to hairpin catalysis.
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ARN Catalítico/química , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , ARN Catalítico/genéticaRESUMEN
Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.