RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
Asunto(s)
SARS-CoV-2 , Humanos , Regulación Alostérica , Secuencia de Aminoácidos , COVID-19 , Microscopía por Crioelectrón , Endorribonucleasas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/químicaRESUMEN
Brazilian plant biodiversity is a rich alternative source of bioactive compounds since plant-derived extracts and/or their secondary metabolites exhibit potential properties to treat several diseases. In this context, Licania rigida Benth (Chrysobalanaceae Family), a large evergreen tree distributed in Brazilian semi-arid regions, deserves attention for its widespread use in popular medicine, although its biological properties are still poorly studied. The aim of this study was to examine (1) acute and sub-chronic oral toxicity at 2000 mg/kg dose; (2) in vitro cytotoxicity at 0.1; 1; 10; 100 or 1000 µg/ml; (3) in vivo mutagenicity at 5, 10 or 20 mg/ml, and (4) potential antioxidant protective effect of L. rigida aqueous leaf extract of (AELr). No marked apparent toxic and genotoxic effects were observed using in vitro and in vivo assays after in vitro treatment of Chinese hamster ovary cell line (CHO-K1) with AELr or in vivo exposure of Wistar rats and Drosophila melanogaster to different extract concentrations. Concerning the antioxidant effect, the extract exhibited a protective effect by decreasing lipid peroxidation as determined by malondialdehyde levels. No significant changes were observed for glutathione (GSH) levels and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Data demonstrate the beneficial potential of AELr to be employed for therapeutic purposes. However, further studies are required to validate the pharmacological application of this plant extract to develop as a phytotherapeutic formulation.
Asunto(s)
Chrysobalanaceae/química , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Animales , Brasil , Células CHO , Cricetulus , Drosophila melanogaster , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Hojas de la Planta/química , Plantas Medicinales/toxicidad , Ratas WistarRESUMEN
Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
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Descubrimiento de Drogas/métodos , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Schistosoma mansoni/enzimología , Tiazoles/metabolismo , Animales , Dominio Catalítico , Cristalización , Cristalografía por Rayos X/métodos , Dimetilsulfóxido/farmacología , Evaluación Preclínica de Medicamentos/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Purina-Nucleósido Fosforilasa/genética , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitologíaRESUMEN
Septins are GTP-binding proteins that will often spontaneously assemble into filaments. In some species, particularly budding yeast, it is well known that these are capable of associating with membranes in order to fulfill their cellular role as a component of the cytoskeleton. Different from other human septins, SEPT7 appears to be unique in that it is an essential component of all hetero-oligomeric complexes described to date. As a step towards understanding the molecular basis of filament assembly, here we present two high-resolution structures of the SEPT7 GTPase domain complexed with GDP. One of these reveals a previously unreported coordination for the magnesium ion involving four water molecules and only a tenuous connection to the protein. The higher resolution structures provide unambiguous insight into the interactions at the G-interface where a structural motif based on an antiparallel ß-bridge allows for the rationalization of why some septins show nucleotide-dependent ß-strand slippage and others do not.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al GTP/química , Septinas/química , Sitios de Unión , Cristalografía por Rayos X , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Manganeso/química , Conformación Proteica en Lámina beta , Dominios Proteicos , Agua/químicaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the ß8-ß9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.
Asunto(s)
Acetilcolina/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolina/química , Regulación Alostérica , Sitio Alostérico , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Dominios Proteicos , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Caracoles , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.
Asunto(s)
Complejos de ATP Sintetasa/metabolismo , Evolución Molecular , Complejos de ATP Sintetasa/química , Complejos de ATP Sintetasa/genética , Secuencia de Aminoácidos , Euryarchaeota/enzimología , Fructosafosfatos/metabolismo , Glucosa/metabolismo , Cinética , Modelos Moleculares , Mutación , Filogenia , Conformación Proteica , Especificidad por SustratoRESUMEN
Septins are filament-forming GTP-binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self-assemble into heterocomplexes, which polymerize into highly organized, cell membrane-interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms. This makes the single septin from Chlamydomonas (CrSEPT) a particularly attractive model for investigating whether functional homopolymeric septin filaments also exist. CrSEPT was detected at the base of the flagella in Chlamydomonas, suggesting that CrSEPT is involved in the formation of a membrane-diffusion barrier. Using transmission electron microscopy, we observed that recombinant CrSEPT forms long filaments with dimensions comparable with those of the canonical structure described for opisthokonts. The GTP-binding domain of CrSEPT purified as a nucleotide-free monomer that hydrolyzes GTP and readily binds its analog guanosine 5'-3-O-(thio)triphosphate. We also found that upon nucleotide binding, CrSEPT formed dimers that were stabilized by an interface involving the ligand (G-interface). Across this interface, one monomer supplied a catalytic arginine to the opposing subunit, greatly accelerating the rate of GTP hydrolysis. This is the first report of an arginine finger observed in a septin and suggests that CrSEPT may act as its own GTP-activating protein. The finger is conserved in all algal septin sequences, suggesting a possible correlation between the ability to form homopolymeric filaments and the accelerated rate of hydrolysis that it provides.
Asunto(s)
Chlamydomonas reinhardtii/química , Complejos Multiproteicos/química , Proteínas de Plantas/química , Multimerización de Proteína , Septinas/química , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Septinas/genética , Septinas/metabolismoRESUMEN
Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named "pathogenicity, virulence and adaptation". One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fosfoglucomutasa/química , Fosfoglucomutasa/metabolismo , Xanthomonas/enzimología , Proteínas Bacterianas/genética , Dominio Catalítico , Citrus/microbiología , Clonación Molecular , Cristalografía por Rayos X , Genes Bacterianos , Cinética , Modelos Moleculares , Mutación , Fosfoglucomutasa/genética , Enfermedades de las Plantas/microbiología , Polisacáridos Bacterianos/biosíntesis , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virulencia/genética , Xanthomonas/genética , Xanthomonas/patogenicidadRESUMEN
Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation, and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins (1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined. Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies. Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin filaments.
Asunto(s)
Schistosoma mansoni/química , Septinas/química , Animales , Sitios de Unión , Calorimetría , Catálisis , Membrana Celular/metabolismo , Cristalografía por Rayos X , Escherichia coli/metabolismo , GTP Fosfohidrolasas/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Hidrólisis , Magnesio/química , Espectroscopía de Resonancia Magnética , Nucleótidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Termodinámica , Agua/químicaRESUMEN
The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.
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Hemoglobinas/química , Oligoquetos/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE: Because most publications on growth and development deal with children with zinc deficiency, we decided to study the effects of this micronutrient on the secretion of growth hormone (GH), insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3), osteocalcin (OCN), and alkaline phosphatase (ALP) in healthy and eutrophic children. This study is original because the methodology was unique. METHODS: Forty schoolchildren participated in the study, 17 females and 23 males, aged 8 and 9 years. The study was carried out during a 3-month period. It was characterized as a triple-blind randomized controlled trial. The children were divided in a control group (20 schoolchildren using 10% sorbitol) and experimental group (20 schoolchildren using zinc). All were submitted to oral zinc supplementation (10 mg Zn/day) and venous zinc administration (0.06537 mg Zn/kg of body weight). Blood samples were collected at 0, 60, 120, 180, and 210 min. All schoolchildren were also submitted to anthropometric, clinical, and dietetic assessments as well as biochemistry analyses. RESULTS: Oral zinc supplementation in the experimental group (1) stimulated an increase in the consumption of protein and fat (p = 0.0007, p < 0.0001, p < 0.0001, respectively), (2) increased basal serum zinc (p < 0.0001), (3) increased plasma ALP (p = 0.0270), and (4) showed a positive correlation for IGF1, IGFBP3, and OCN, comparing before and after oral zinc supplementation (p = 0.0011, p < 0.0001, p < 0.0446, respectively). During zinc administration, plasma IGF1 and IGFBP3 increased significantly in the experimental group (p = 0.0468, p < 0.0001, respectively). Plasma GH increased in the experimental group but without statistical difference comparing before and after oral zinc supplementation. CONCLUSIONS: Zinc supplementation stimulated an increase in the consumption of some macronutrients and basal serum zinc and improved plasma alkaline phosphatase levels. Zinc administration increased hormones of the GH-IGF1 system.
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Fosfatasa Alcalina/sangre , Suplementos Dietéticos , Hormona de Crecimiento Humana/sangre , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Osteocalcina/sangre , Zinc/farmacología , Niño , Femenino , Voluntarios Sanos , Humanos , Masculino , Valores de Referencia , Oligoelementos/farmacología , Zinc/sangreRESUMEN
BACKGROUND/AIMS: Berardinelli-Seip syndrome (BSS) is a recessive autosomal genetic disorder characterized by the near loss of adipose tissue with disturbance in lipid metabolism. METHODS: Biochemical and hormonal parameters and Pro12Ala, Pvull, Avall, Sstl and ADIPOQ polymorphisms in 22 patients with BSS were analyzed and examined for a possible association with lipid profiles. RESULTS: Parental consanguinity, insulin resistance and diabetes mellitus were observed in 63.6, 81.8 and 59.1% of patients, respectively. All individuals presented high triglyceride levels, and 68.1% of patients showed high cholesterol levels. The Pro/Pro genotype of the Pro12Ala polymorphism of the PPARγ2 gene was found in 86.3% of patients; the Ala/Ala variant was not observed in any patient. The PvuII polymorphism of the LPL gene showed a frequency of 50% for the P1P2 variant. The AvaII polymorphism of the LDLR gene showed a similar frequency of 40.9% for both CT and TT variants. The S1S1 genotype of the Sstl polymorphism of the APOC3 gene had a frequency of 86.3%. The CC allele of the ADIPOQ polymorphism of the adiponectin gene was found in 54.6% of patients. CONCLUSIONS: No association was found between lipid parameters and the relevant Pvull, Avall and Sstl polymorphisms. However, we did observe an association of the Pro12Ala and ADIPOQ polymorphisms with higher lipid levels, suggesting a close relationship between these factors.
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Adiponectina/genética , Predisposición Genética a la Enfermedad , Lipodistrofia Generalizada Congénita/genética , PPAR gamma/genética , Adulto , Brasil , Colesterol/sangre , Femenino , Humanos , Lipodistrofia Generalizada Congénita/sangre , Masculino , Polimorfismo de Nucleótido Simple , Triglicéridos/sangre , Población BlancaRESUMEN
INTRODUCTION: The thyroid cancer has the ninth larger incidence of cancer in the world. Investigations related to the exposure to metals have become important due to the sensibility of the thyroid gland to them. Studies reveal that carcinogenic progressions are associated to the deficiency of the essential trace elements. In this context, the zinc is highlighted, essential for the metabolism of the thyroidal hormone and has a potential relation with the pathogenesis of the thyroid cancer. The objective of this systematic review and meta-analysis is to evaluate the low serum zinc as a risk factor for thyroid cancer in adults. METHODS AND ANALYSIS: PubMed/MEDLINE, Scopus, Embase and LILACS databases will be searched for observational studies investigating the low serum zinc as a risk factor for thyroid cancer in adults. No language or publication period restrictions will be imposed. The primary outcome will be that the low serum zinc is a risk factor for thyroid cancer. Three independent reviewers will select the studies and extract data from the original publications. The risk-of-bias will be assessed by using the Newcastle-Ottawa Quality Assessment Scale (NOS). Data synthesis will be performed using the R software (V.4.3.1) and to assess heterogeneity, we will compute the I2 statistic and the results will be based on either random-effects or fixed-effects models, depending on the heterogeneity. The Grading of Recommendations, Development, and Evaluation (GRADE) system will be used to evaluate the reliability and quality of evidence. PROSPERO REGISTRATION NUMBER: International Prospective Register of Systematic Reviews (PROSPERO) CRD42023463747.
Asunto(s)
Metaanálisis como Asunto , Revisiones Sistemáticas como Asunto , Neoplasias de la Tiroides , Zinc , Humanos , Neoplasias de la Tiroides/sangre , Neoplasias de la Tiroides/epidemiología , Zinc/sangre , Factores de RiesgoRESUMEN
BACKGROUND & AIMS: Duchenne muscular dystrophy (DMD) involves muscle fragility, sarcolemma instability, and chronic inflammation. This study aims to identify the inflammatory profile of DMD patients and evaluate associations between clinical and nutritional variables. METHODS: We performed a cross-sectional study nested in a cohort to obtain sociodemographics, illness time, use of medications, and supplement data through interviews and the patient's medical records. Then, we assessed the relationships between illness time, cytokine levels, and nutritional status. RESULTS: Forty-four male participants, aged 4.3-24.2 years, were evaluated. Concerning nutritional status, 18 participants were eutrophic. The fat mass increased and the lean mass decreased from the beginning of the first signs of DMD. Cytokines levels in DMD patients, even under corticosteroids therapy, are higher than values described in the literature on healthy subjects. The regression models demonstrated that illness time and BMI/A z-scores are associated with higher values of interleukin-6. CONCLUSIONS: A persistent inflammatory profile was observed in the patients evaluated. The data suggest that maintaining adequate nutritional status and body composition is important for determining the inflammation presented by individuals with DMD.
Asunto(s)
Composición Corporal , Inflamación , Distrofia Muscular de Duchenne , Estado Nutricional , Humanos , Distrofia Muscular de Duchenne/complicaciones , Masculino , Estudios Transversales , Adolescente , Niño , Adulto Joven , Preescolar , Citocinas/sangre , Índice de Masa Corporal , Interleucina-6/sangreRESUMEN
Schistosomiasis is caused by parasites of the genus Schistosoma, which infect more than 200 million people. Praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, but despite that it is ineffective against juvenile worms and size and taste issues with its pharmaceutical forms impose challenges for treating school-aged children. It is also important to note that PZQ resistant strains can be generated in laboratory conditions and observed in the field, hence its extensive use in mass drug administration programs raises concerns about resistance, highlighting the need to search for new schistosomicidal drugs. Schistosomes survival relies on the redox enzyme thioredoxin glutathione reductase (TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a high-throughput fragment screening campaign of 768 compounds against S. mansoni TGR (SmTGR) using X-ray crystallography. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within SmTGR, a noteworthy exception being the "doorstop pocket" near the NADPH binding site. We have compared results from hotspots and pocket druggability analysis of SmTGR with the experimental binding sites found in this work, with our results indicating only limited coincidence between experimental and computational results. Finally, we discuss that binding sites at the doorstop/NADPH binding site and in the SmTGR dimer interface, should be prioritized for developing SmTGR inhibitors as new antischistosomal drugs.
Asunto(s)
Complejos Multienzimáticos , NADH NADPH Oxidorreductasas , Esquistosomiasis mansoni , Esquistosomiasis , Animales , Niño , Humanos , Schistosoma mansoni , Cristalografía por Rayos X , NADP/metabolismo , Esquistosomiasis/tratamiento farmacológico , Sitios de Unión , Esquistosomiasis mansoni/parasitologíaRESUMEN
Crystal dehydration is a post-crystallization technique that can potentially improve the diffraction of macromolecular crystals. There are currently several ways of undertaking this process; however, dehydration experiments are often limited in their throughput and require prior manipulation of the samples. In the present study, a novel method is proposed that uses in situ plate screening to assess the effect of dehydration by combining the throughput of 96-well crystallization plates with direct X-ray feedback on crystal diffraction quality.
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Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas/química , Cristalización/instrumentación , Cristalización/métodos , Cristalografía por Rayos XRESUMEN
In adult schistosomes, the enzyme adenosine kinase (AK) is responsible for the incorporation of some adenosine analogues, such as 2-fluoroadenosine and tubercidin, into the nucleotide pool, but not others. In the present study, the structures of four complexes of Schistosoma mansoni AK bound to adenosine and adenosine analogues are reported which shed light on this observation. Two differences in the adenosine-binding site in comparison with the human counterpart (I38Q and T36A) are responsible for their differential specificities towards adenosine analogues, in which the Schistosoma enzyme does not tolerate bulky substituents at the N7 base position. This aids in explaining experimental data which were reported in the literature more than two decades ago. Furthermore, there appears to be considerable plasticity within the substrate-binding sites that affects the side-chain conformation of Ile38 and causes a previously unobserved flexibility within the loop comprising residues 286-299. These results reveal that the latter can be sterically occluded in the absence of ATP. Overall, these results contribute to the body of knowledge concerning the enzymes of the purine salvage pathway in this important human parasite.
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Adenosina Quinasa/química , Adenosina/química , Schistosoma mansoni/enzimología , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina Quinasa/genética , Adenosina Quinasa/metabolismo , Animales , Cristalización , Cristalografía por Rayos X , Humanos , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.