RESUMEN
Schistosoma japonicum glutathione transferase (Sj26GST), an enzyme central to detoxification of electrophilic compounds in the parasite, is upregulated in response to drug treatment. Therefore, Sj26GST may serve as a potential therapeutic target for the treatment of schistosomiasis. Herewith, we describe the structural basis of inhibition of Sj26GST by ellagic acid (EA). Using 1-chloro-2,4-dinitrobenzene and reduced glutathione (GSH) as Sj26GST substrates, EA was shown to inhibit Sj26GST activity by 66 % with an IC50 of 2.4 µM. Fluorescence spectroscopy showed that EA altered the polarity of the environment of intrinsic tryptophan and that EA decreased (in a dose-dependent manner) the interaction between Sj26GST and 8-Anilino-1-naphthalenesulfonate (ANS), which is a known GST H-site ligand. Thermodynamic studies indicated that the interaction between Sj26GST and EA is spontaneous (ΔG = -29.88 ± 0.07 kJ/mol), enthalpically-driven (ΔH = -9.48 ± 0.42 kJ/mol) with a favourable entropic change (ΔS = 20.40 ± 0.08 kJ/mol/K), and with a stoichiometry of four EA molecules bound per Sj26GST dimer. The 1.53 Å-resolution Sj26GST crystal structure (P 21 21 21 space group) complexed with GSH and EA shows that EA binds primarily at the dimer interface, stabilised largely by Van der Waal forces and H-bonding. Besides, EA bound near the H-site and less than 3.5 Å from the ε-NH2 of the γ-glutamyl moiety of GSH, in each subunit.
Asunto(s)
Inhibidores Enzimáticos/química , Glutatión Transferasa/química , Proteínas del Helminto/química , Schistosoma japonicum/enzimología , Animales , Calorimetría , Fenómenos Químicos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Proteínas del Helminto/antagonistas & inhibidores , Cinética , Ligandos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Unión Proteica , Schistosoma japonicum/efectos de los fármacos , Relación Estructura-Actividad , TermodinámicaRESUMEN
BACKGROUND: Protease inhibitors form the main component of second-line antiretroviral treatment in South Africa. Despite their efficacy, mutations arising within the HIV-1 gag and protease coding regions contribute to the development of resistance against this class of drug. In this paper we investigate a South African HIV-1 subtype C Gag-protease that contains a hinge region mutation and insertion (N37T↑V). METHODS: In vitro single-cycle drug susceptibility and viral replication capacity assays were performed on W1201i, a wild-type reference isolate (MJ4) and a chimeric construct (MJ4GagN37T↑VPR). Additionally, enzyme assays were performed on the N37T↑V protease and a wild-type reference protease. RESULTS: W1201i showed a small (threefold), but significant (P<0.0001) reduction in drug susceptibility to darunavir compared with MJ4. Substitution of W1201i-Gag with MJ4-Gag resulted in an additional small (twofold), but significant (P<0.01) reduction in susceptibility to lopinavir and atazanavir. The W1201i pseudovirus had a significantly (P<0.01) reduced replication capacity (16.4%) compared with the MJ4. However, this was dramatically increased to 164% (P<0.05) when W1201i-Gag was substituted with MJ4-Gag. Furthermore, the N37T↑V protease displayed reduced catalytic processing compared with the SK154 protease. CONCLUSIONS: Collectively, these data suggest that the N37T↑V mutation and insertion increases viral infectivity and decreases drug susceptibility. These variations are classified as secondary mutations, and indirectly impact inhibitor binding, enzyme fitness and enzyme stability. Additionally, polymorphisms arising in Gag can modify the impact of protease with regards to viral replication and susceptibility to protease inhibitors.
Asunto(s)
Farmacorresistencia Viral , Variación Genética , Genotipo , Infecciones por VIH/virología , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/fisiología , Replicación Viral , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/química , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Fenotipo , Relación Estructura-ActividadRESUMEN
HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.
Asunto(s)
Darunavir/química , Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1 , Lopinavir/química , Mutagénesis Insercional , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Darunavir/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Proteasa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Humanos , Lopinavir/farmacología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides 99EIWD102 and 92ATSPYT97 found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "158SWNSGALTGHTFPAVL175" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "4LQESGSVL11 from the heavy chain and 4LTQSPSSL11 from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Virus de la Rabia/inmunología , Rabia/terapia , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Anticuerpos Antivirales/uso terapéutico , Frío/efectos adversos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Simulación por Computador , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Pruebas de Neutralización , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos , Proteolisis , Rabia/inmunología , Rabia/virología , Especies Reactivas de Oxígeno/química , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
FOXP2 is a transcriptional repressor involved in development of the human brain and is the first gene product to be linked to the evolution of human speech. FOXP2 belongs to the FOX superfamily of proteins that share a common winged helix DNA binding domain - the forkhead domain. A divalent cation (Mg2+ or Ca2+) has been identified bound to a group of highly conserved residues in a number of FOX forkhead domain crystal structures. This work aims to investigate the role of the conserved divalent cation binding site by studying both the structure and DNA-binding function of the FOXP2 forkhead domain when in the presence and absence of either cation (Mg2+or Ca2+). The presence of the cations does not significantly alter the structure of the apo-FOXP2 forkhead domain. However, when in the presence of a cognate oligonucleotide sequence, differences are observed upon addition of divalent cation. These differences occur both in the structure and in the thermodynamic DNA binding signature of the FOXP2 forkhead domain. The incorporation of molecular dynamics simulations together with the experimental data provides us with sufficient insight so as to propose a possible role for divalent cations in the regulation of DNA binding to FOX transcription factors.
Asunto(s)
Calcio/metabolismo , ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Magnesio/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Escherichia coli/genética , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Alineación de Secuencia , TermodinámicaRESUMEN
Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70-x (PfHsp70-x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite-infected red blood cell (RBC) along with PfHsp70-x. The role of PfHsp70-x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70-x is the presence of EEVN residues at its C-terminus. In this regard, PfHsp70-x resembles canonical eukaryotic cytosol-localized Hsp70s which possess EEVD residues at their C-termini in place of the EEVN residues associated with PfHsp70-x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co-chaperones. Characterization of the role of the EEVN residues of PfHsp70-x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70-x (full length) and its EEVN minus form (PfHsp70-xT ). We then conducted structure- function assays towards establishing the role of the EEVN motif of PfHsp70-x. Our findings suggest that the EEVN residues of PfHsp70-x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70-x and human Hsp70-Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70-x and hHsp90 cooperate in vivo.
Asunto(s)
Proteínas HSP70 de Choque Térmico/química , Malaria Falciparum/parasitología , Plasmodium falciparum/química , Proteínas Protozoarias/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Malaria Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Unión Proteica , Pliegue de Proteína , Mapas de Interacción de Proteínas , Proteínas Protozoarias/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression of large quantities is prevented due to the enzyme's autolytic nature and its inherent cytotoxicity in Escherichia coli cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin-hexahistidine fusion system for the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wild-type protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage of the thioredoxin-hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after cleavage of the tag. The novel thioredoxin-hexahistidine fusion system, therefore, enables the successful overexpression and purification of catalytically active HIV-1 proteases.
Asunto(s)
Cromatografía de Afinidad/métodos , Variación Genética , Proteasa del VIH/clasificación , Proteasa del VIH/metabolismo , VIH-1/enzimología , Histidina/química , Oligopéptidos/química , Proteínas Recombinantes de Fusión/metabolismo , Tiorredoxinas/química , Secuencia de Aminoácidos , Proteasa del VIH/genética , Proteasa del VIH/aislamiento & purificación , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de SecuenciaRESUMEN
Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.
Asunto(s)
ADN/química , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Transcripción Genética , Sitios de Unión , ADN/metabolismo , HumanosRESUMEN
FOXP2 is a transcription factor expressed in multiple tissues during embryonic development. FOXP2 regulates transcription by binding to DNA at its DNA binding domain, the forkhead domain (FHD) through the recognition helix. Ser557 is a residue located within the recognition helix that has the potential to become phosphorylated posttranslationally. In this study we investigated whether phosphorylation of Ser557 can influence the structure and DNA binding of the FOXP2 FHD. We did this by constructing S557E, a phosphomimetic mutant, and comparing its behaviour to the wild type. The mutation did not affect the secondary or tertiary structure of the protein although it did decrease the propensity of the FOXP2 FHD to form dimers. Most notably, the mutation showed significantly reduced DNA binding compared to the wild type as detected using electrophoretic mobility shift assays. Molecular docking was also performed in which the wild type, phosphomimetic mutant and phosphorylated wild-type were docked to DNA and their interactions with DNA were compared. These results indicated that the wild type forms more interactions with the DNA and that the phosphomimetic mutant as well as the phosphorylated wild type did not associate as favourably with the DNA. This indicates that phosphorylation of Ser557 could disrupt DNA binding likely due to electrostatic and steric hindrance. This suggests that phosphorylation of Ser557 in the FOXP2 FHD could act as a control mechanism for FOXP2 and ultimately could be involved in regulation of transcription.
Asunto(s)
ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Mutación , Serina/metabolismo , ADN/química , ADN/genética , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Humanos , Simulación del Acoplamiento Molecular , Imitación Molecular , Fosforilación , Conformación Proteica , Dominios Proteicos , Serina/química , Serina/genéticaRESUMEN
HIV-1 protease is an important antiretroviral drug target due to its key role in viral maturation. Computational models have been successfully used in the past to understand the dynamics of HIV-1 protease variants. We performed molecular dynamics simulations and induced fit docking on a wild-type South African HIV-1 subtype C protease and an N37T↑V hinge region variant. The simulations were initiated in a cubic cell universe and run in explicit solvent, with the wild-type and variant proteases in the fully closed conformation and under periodic boundary conditions. The trajectory for each simulation totalled 20â¯ns. The results indicate that the N37T↑V hinge region mutation and insertion alter the molecular dynamics of the flap and hinge regions when compared to the wild-type protease. Specifically, the destabilisation of the hinge region allowed a larger and protracted opening of the flap region due to the formation of two key hinge/cantilever salt-bridges, which are absent in the wild-type protease. Domain-domain anti-correlation was observed between the flap and hinge region for both models. However, the N37T↑V variant protease displayed a lower degree of anti-correlation. The mutations affected the thermodynamic landscape of inhibitor binding as there were fewer observable chemical contacts between the N37T↑V variant protease and lopinavir, atazanavir and darunavir, respectively. These data elucidate the biophysical basis for the selection of hinge region insertion mutations by the HI virus.
Asunto(s)
Variación Genética , Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Ligandos , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad Cuantitativa , SudáfricaRESUMEN
The ß-subunit of the human eukaryotic elongation factor 1 complex (heEF1ß) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α- and γ-subunits (eEF1γ). To biophysically characterize heEF1ß, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL-heEF1ß), N-terminus (NT-heEF1ß), and the C-terminus (CT-heEF1ß) regions of the protein. Our results suggest that heEF1ß is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1ß. Both FL-heEF1ß and NT-heEF1ß form dimers of size 62 and 30 kDa, respectively, but the CT-heEF1ß is monomeric. FL-heEF1ß interacts with the N-terminus glutathione transferase-like domain of heEF1γ (NT-heEF1γ) to form a 195-kDa complex or a 230-kDa complex in the presence of oxidized glutathione. On the other hand, NT-heEF1ß forms a 170-kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL-heEF1ß associated with monomeric or dimeric NT-heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (KD = 9.6 nM for monomeric or 11.3 nM for dimeric NT-heEF1γ). We postulate that the N-terminus region of heEF1ß may be responsible for its dimerization and the C-terminus region of heEF1ß modulates the formation of an ordered heEF1ß-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.
Asunto(s)
Glutatión/química , Factor 1 de Elongación Peptídica/química , Subunidades de Proteína/química , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutatión/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-androstene-3-17-dione. GSTA3-3 catalyzes this reaction with ten-fold greater efficiency than GSTA1-1, its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable, subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations, singly or together, have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyze their reactions. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously known.
Asunto(s)
Arginina/genética , Glutatión Transferasa/química , Tirosina/genética , Catálisis , Dominio Catalítico , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Simulación de Dinámica Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estabilidad Proteica , Especificidad por SustratoRESUMEN
The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 µM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.
Asunto(s)
Disulfuro de Glutatión/química , Glutatión/análogos & derivados , Glutatión/química , Factor 1 de Elongación Peptídica/química , Calorimetría , Expresión Génica , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Ligandos , Simulación de Dinámica Molecular , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Temperatura , TermodinámicaRESUMEN
Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members.
Asunto(s)
Reacciones Cruzadas/inmunología , Glicoproteínas/inmunología , Lyssavirus/inmunología , Lyssavirus/metabolismo , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/virología , Proteínas Virales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Secuencia Conservada , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Glicoproteínas/química , Glicoproteínas/genética , Lyssavirus/clasificación , Lyssavirus/genética , Modelos Moleculares , Pruebas de Neutralización , Filogenia , Conformación Proteica , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.
Asunto(s)
Proteínas del Choque Térmico HSP110/genética , Proteínas del Choque Térmico HSP72/metabolismo , Malaria Falciparum/genética , Plasmodium falciparum/genética , Adenosina Trifosfatasas/genética , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Malaria Falciparum/parasitología , Chaperonas Moleculares , Nucleótidos/genética , Plasmodium falciparum/patogenicidad , Dominios Proteicos/genética , Pliegue de ProteínaRESUMEN
Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Adenosina Trifosfatasas/metabolismo , Expresión Génica , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Respuesta al Choque Térmico , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1-4) subfamily is unique in the family in that the forkhead domains of these proteins are able to dimerise via domain swapping. In this event, structural elements are exchanged via extension of the hinge loop region. Despite the high sequence homology among the FOXP subfamily members, the stability of their forkhead domain dimers varies, with FOXP3 forming the most stable dimer. An amino acid difference is observed in the hinge region of the FOXP subfamily where a tyrosine in all members is replaced with a phenylalanine in FOXP3. In this work, the role of phenylalanine at this position in the hinge region was investigated. This was done by creating the Y540F variant of the FOXP2 forkhead domain. The effect of the Y540F mutation on the structure, dimerisation propensity and DNA binding ability of the FOXP subfamily was investigated. The mutation altered the structure of the protein by decreasing the disorder of the backbone as measured by circular dichroism spectroscopy and by altering the local environment of the hinge region as measured by tryptophan fluorescence. The propensity of the forkhead domain to form a dimer was improved ~9.5 fold by the mutation. This was attributed to increased hydrophobicity at the dimer interface as well as altered tension in the hinge loop region. DNA binding assays indicated that the affinity for DNA was decreased by the mutation. Taken together, these findings suggest that domain swapping may modulate DNA binding.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Factores de Transcripción Forkhead/química , Multimerización de Proteína , Tirosina/química , Secuencia de Aminoácidos , Cromatografía en Gel , Dicroismo Circular , Cristalografía por Rayos X , ADN/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Factores de Transcripción Forkhead/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Tirosina/genéticaRESUMEN
Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV-1 subtype C (C-SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV-1 subtype B protease. The heavy HIV burden in sub-Saharan Africa, where subtype C HIV-1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C-SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C-SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four- and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C-SA protease were investigated by performing hydrogen-deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C-SA proteases. The reduced propensity to form the E35-R57 salt bridge, and alterations in the hydrophobic core of the C-SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N-terminus of both proteases is less stable than the C-terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C-terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.
Asunto(s)
Amidas/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Aminoácidos , Dimerización , Proteasa del VIH/química , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen-deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1ß1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1ß1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1ß1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1ß1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1ß1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1ß1 is regulated.
Asunto(s)
Escherichia coli/enzimología , Hidrógeno/química , Proteína Quinasa 10 Activada por Mitógenos/química , Proteína Quinasa 8 Activada por Mitógenos/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Medición de Intercambio de Deuterio , Activación Enzimática , Humanos , Sistema de Señalización de MAP Quinasas , Espectrometría de Masas , Datos de Secuencia Molecular , Nucleótidos/química , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Solventes/química , Temperatura , Termodinámica , Urea/químicaRESUMEN
The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.