RESUMEN
The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -Éßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal É-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.
Asunto(s)
Acinetobacter baumannii , ATPasas de Translocación de Protón , Humanos , ATPasas de Translocación de Protón/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Hidrólisis , Microscopía por Crioelectrón , Secuencia de Aminoácidos , Bacterias/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium , Tuberculosis , Humanos , Micobacterias no Tuberculosas , Hidrólisis , Adenosina TrifosfatoRESUMEN
Mycobacterium tuberculosis (Mtb) aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure-function analyses we previously identified POA-based inhibitors of Mtb PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure-function studies on PanD encoded by the nontuberculous mycobacterial lung pathogen Mycobacterium abscessus (Mab), shedding light on the differences and similarities of Mab and Mtb PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric Mab PanD, which in comparison to the structure of the derived C-terminal truncated Mab PanD1-114 mutant revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of Mab PanD1-114 explored the essentiality of the C-terminus for catalysis. A library of recombinant Mab PanD mutants based on structural information and PZA/POA resistant PanD mutations in Mtb illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogues, we identified (3-(1-naphthamido)pyrazine-2-carboxylic acid) (analogue 2) as the first potent inhibitor of Mab PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analogue 2 to Mab PanD. In contrast to PZA and POA, which are whole-cell inactive, analogue 2 exerts appreciable antibacterial activity against the three subspecies of Mab.
Asunto(s)
Mycobacterium abscessus , Pirazinamida , Antituberculosos/farmacología , Carboxiliasas , Pirazinamida/análogos & derivados , Pirazinamida/farmacologíaRESUMEN
While many bacteria are able to bypass the requirement for oxidative phosphorylation when grown on carbohydrates, Mycobacterium tuberculosis is unable to do so. Differences of amino acid composition and structural features of the mycobacterial F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) compared to its prokaryotic or human counterparts were recently elucidated and paved avenues for the discovery of molecules interfering with various regulative mechanisms of this essential energy converter. In this context, the mycobacterial peripheral stalk subunit δ came into focus, which displays a unique N-terminal 111-amino acid extension. Here, mutants of recombinant mycobacterial subunit δ were characterized, revealing significant reduction in ATP synthesis and demonstrating essentiality of this subunit for effective catalysis. These results provided the basis for the generation of a four-feature model forming a δ receptor-based pharmacophore and to identify a potent subunit δ inhibitor DeMF1 via in silico screening. The successful targeting of the δ subunit demonstrates the potential to advance δ's flexible coupling as a new area for the development of F-ATP synthase inhibitors.
Asunto(s)
Mycobacterium tuberculosis , ATPasas de Translocación de Protón , Adenosina Trifosfato/metabolismo , Aminoácidos/farmacología , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/genéticaRESUMEN
Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Celulosa/metabolismo , Glucosiltransferasas/química , Uridina Difosfato Glucosa/química , Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Manganeso/química , Manganeso/metabolismo , Mutación , Multimerización de Proteína , Uridina Difosfato Glucosa/metabolismoRESUMEN
In contrast to most bacteria, the mycobacterial F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F1 -ATPase component α3 :ß3 :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1 -ATPase (MsF1 -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF1 αßγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF1 αßγεΔ2-5 , revealed similar ATP hydrolysis as MsF1 αßγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF1 αßγεΔ121 and MsF1 αßγεΔ103-121 with deletion of the C-terminal residue D121 and the two C-terminal É-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3 ß3 -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/enzimología , ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidrólisis/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Mutación , Mycobacterium , Mycobacterium smegmatis/genética , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The F1 FO -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , ATPasas de Translocación de Protón Bacterianas/antagonistas & inhibidores , Diarilquinolinas/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Benzamidas/química , Benzamidas/farmacología , Benzamidas/toxicidad , Sinergismo Farmacológico , Células Madre Embrionarias/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/toxicidad , Relación Estructura-ActividadRESUMEN
The causative agent of Tuberculosis (TB) Mycobacterium tuberculosis (Mtb) encounters unfavourable environmental conditions in the lungs, including nutrient limitation, low oxygen tensions and/or low/high pH values. These harsh conditions in the host triggers Mtb to enter a dormant state in which the pathogen does not replicate and uses host-derived fatty acids instead of carbohydrates as an energy source. Independent to the energy source, the bacterium's energy currency ATP is generated by oxidative phosphorylation, in which the F1FO-ATP synthase uses the proton motive force generated by the electron transport chain. This catalyst is essential in Mtb and inhibition by the diarylquinoline class of drugs like Bedaquilline, TBAJ-587, TBAJ-876 or squaramides demonstrated that this engine is an attractive target in TB drug discovery. A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its latent ATPase activity, to prevent energy waste and to control the membrane potential. Recently, unique epitopes of mycobacterial F1FO-ATP synthase subunits absent in their prokaryotic or mitochondrial counterparts have been identified to contribute to the regulation of the low ATPase activity. Most recent structural insights into individual subunits, the F1 domain or the entire mycobacterial enzyme added to the understanding of mechanisms, regulation and differences of the mycobacterial F1FO-ATP synthase compared to other bacterial and eukaryotic engines. These novel insights provide the basis for the design of new compounds targeting this engine and even novel regimens for multidrug resistant TB.
Asunto(s)
Antituberculosos/metabolismo , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Diseño de Fármacos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The F-ATP synthase is an essential enzyme in mycobacteria, including the pathogenic Mycobacterium tuberculosis. Several new compounds in the TB-drug pipeline target the F-ATP synthase. In light of the importance and pharmacological attractiveness of this novel antibiotic target, tools have to be developed to generate a recombinant mycobacterial F1FO ATP synthase to achieve atomic insight and mutants for mechanistic and regulatory understanding as well as structure-based drug design. Here, we report the first genetically engineered, purified and enzymatically active recombinant M. smegmatis F1FO ATP synthase. The projected 2D- and 3D structures of the recombinant enzyme derived from negatively stained electron micrographs are presented. Furthermore, the first 2D projections from cryo-electron images are revealed, paving the way for an atomic resolution structure determination.
Asunto(s)
ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes/metabolismo , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Hidrólisis , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , ATPasas de Translocación de Protón/aislamiento & purificación , ATPasas de Translocación de Protón/ultraestructura , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The dynamic interaction of the N- and C-terminal domains of mycobacterial F-ATP synthase subunit ε is proposed to contribute to efficient coupling of H+-translocation and ATP synthesis. Here, we investigate crosstalk between both subunit ε domains by introducing chromosomal atpC missense mutations in the C-terminal helix 2 of ε predicted to disrupt inter domain and subunit ε-α crosstalk and therefore coupling. The ε mutant εR105A,R111A,R113A,R115A (ε4A) showed decreased intracellular ATP, slower growth rates and lower molar growth yields on non-fermentable carbon sources. Cellular respiration and metabolism were all accelerated in the mutant strain indicative of dysregulated oxidative phosphorylation. The ε4A mutant exhibited an altered colony morphology and was hypersusceptible to cell wall-acting antimicrobials suggesting defective cell wall biosynthesis. In silico screening identified a novel mycobacterial F-ATP synthase inhibitor disrupting ε's coupling activity demonstrating the potential to advance this regulation as a new area for mycobacterial F-ATP synthase inhibitor development.
Asunto(s)
Adenosina Trifosfato/metabolismo , Mutación , Mycobacterium/crecimiento & desarrollo , ATPasas de Translocación de Protón/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular , Simulación por Computador , Metabolismo Energético , Modelos Moleculares , Mycobacterium/enzimología , Mycobacterium/genética , Conformación Proteica , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genéticaRESUMEN
Zika virus (ZIKV) relies on its nonstructural protein 5 (NS5) for capping and synthesis of the viral RNA. Recent small-angle X-ray scattering (SAXS) data of recombinant ZIKV NS5 protein showed that it is dimeric in solution. Here, we present insights into the critical residues responsible for its dimer formation. SAXS studies of the engineered ZIKV NS5 mutants revealed that R681A mutation on NS5 (NS5R681A ) disrupts the dimer formation and affects its RNA-dependent RNA polymerase activity as well as the subcellular localization of NS5R681A in mammalian cells. The critical residues involved in the dimer arrangement of ZIKV NS5 are discussed, and the data provide further insights into the diversity of flaviviral NS5 proteins in terms of their propensity for oligomerization.
Asunto(s)
Proteínas no Estructurales Virales/metabolismo , Virus Zika/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Dimerización , Humanos , Mutación , Conformación Proteica , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Difracción de Rayos XRESUMEN
The Zika virus (ZIKV) epidemic in the Americas was alarming because of its link with microcephaly in neonates and Guillain-Barré syndrome in adults. The unusual pathologies induced by ZIKV infection and the knowledge that the flaviviral nonstructural protein 5 (NS5), the most conserved protein in the flavivirus proteome, can modulate the host immune response during ZIKV infection prompted us to investigate the subcellular localization of NS5 during ZIKV infection and explore its functional significance. A monopartite nuclear localization signal (NLS) sequence within ZIKV NS5 was predicted by the cNLS Mapper program, and we observed localization of ZIKV NS5 in the nucleus of infected cells by immunostaining with specific antibodies. Strikingly, ZIKV NS5 forms spherical shell-like nuclear bodies that exclude DNA. The putative monopartite NLS 390KRPR393 is necessary to direct FLAG-tagged NS5 to the nucleus as the NS5 390ARPA393 mutant protein accumulates in the cytoplasm. Furthermore, coimmunostaining experiments reveal that NS5 localizes with and sequesters importin-α, but not importin-ß, in the observed nuclear bodies during virus infection. Structural and biochemical data demonstrate binding of ZIKV NS5 with importin-α and reveal important binding determinants required for their interaction and formation of complexes that give rise to the supramolecular nuclear bodies. Significantly, we demonstrate a neuronal-specific activation of the host immune response to ZIKV infection and a possible role of ZIKV NS5's nuclear localization toward this activation. This suggests that ZIKV pathogenesis may arise from a tissue-specific host response to ZIKV infection.
Asunto(s)
Interacciones Microbiota-Huesped/inmunología , Neuronas/inmunología , Neuronas/virología , Proteínas no Estructurales Virales/metabolismo , Virus Zika/inmunología , alfa Carioferinas/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HEK293 , Humanos , Inflamación/genética , Masculino , Ratones , Unión Proteica , Proteínas no Estructurales Virales/genética , Replicación Viral , Virus Zika/genética , Virus Zika/fisiologíaRESUMEN
Dengue- (DENV) and Zika viruses (ZIKV) rely on their non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA, and the non-structural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. Accumulation of data for DENV- and ZIKV NS3 and NS5 in solution during recent years provides information about their overall shape, substrate-induced alterations, oligomeric forms and flexibility, with the latter being essential for domain-domain crosstalk. The importance and differences of the linker regions that connect the two domains of NS3 or NS5 are highlighted in particular with respect to the different DENV serotypes (DENV-1 to -4) as well as to the sequence diversities between the DENV and ZIKV proteins. Novel mutants of the French Polynesia ZIKV NS3 linker presented, identify critical residues in protein stability and enzymatic activity.
Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Estabilidad Proteica , ARN Helicasas/química , ARN Helicasas/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , SolucionesRESUMEN
The 11 kDa, positively charged dengue capsid protein (C protein) exists stably as a homodimer and colocalizes with the viral genome within mature viral particles. Its core is composed of four alpha helices encompassing a small hydrophobic patch that may interact with lipids, but approximately 20% of the protein at the N-terminus is intrinsically disordered, making it challenging to elucidate its conformational landscape. Here, we combine small-angle X-ray scattering (SAXS), amide hydrogen-deuterium exchange mass spectrometry (HDXMS), and atomic-resolution molecular dynamics (MD) simulations to probe the dynamics of dengue C proteins. We show that the use of MD force fields (FFs) optimized for intrinsically disordered proteins (IDPs) is necessary to capture their conformational landscape and validate the computationally generated ensembles with reference to SAXS and HDXMS data. Representative ensembles of the C protein dimer are characterized by alternating, clamp-like exposure and occlusion of the internal hydrophobic patch, as well as by residual helical structure at the disordered N-terminus previously identified as a potential source of autoinhibition. Such dynamics are likely to determine the multifunctionality of the C protein during the flavivirus life cycle and hence impact the design of novel antiviral compounds.
Asunto(s)
Proteínas de la Cápside/química , Virus del Dengue/química , Proteínas Intrínsecamente Desordenadas/química , Espectrometría de Masas , Simulación de Dinámica Molecular , Conformación Proteica , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Despite the highly oxidative environment of the phagosomal lumen, the need for maintaining redox homeostasis is a critical aspect of mycobacterial biology. The pathogens are equipped with the sophisticated thioredoxin- (Trx) and peroxiredoxin system, including TrxC and the alkyl hydroperoxide reductase subunit C (AhpC), whereby TrxC is one of the reducing partners of AhpC. Here we visualize the redox modulated dodecamer ring formation of AhpC from Mycobacterium bovis (BCG strain; MbAhpC) using electron microscopy and present novel insights into the unique N-terminal epitope (40 residues) of mycobacterial AhpC. Truncations and amino acid substitutions of residues in the unique N-terminus of MbAhpC provide insights into their structural and enzymatic roles, and into the evolutionary divergence of mycobacterial AhpC versus that of other bacteria. These structural details shed light on the epitopes and residues of TrxC which contributes to its interaction with AhpC. Since human cells lack AhpC, the unique N-terminal epitope of mycobacterial AhpC as well as the MbAhpC-TrxC interface represent an ideal drug target.
Asunto(s)
Mycobacterium bovis/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dispersión Dinámica de Luz , Epítopos/química , Homeostasis , Peróxido de Hidrógeno/química , Microscopía Electrónica , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mycobacterium bovis/química , Mycobacterium bovis/genética , Oxidación-Reducción , Peroxirredoxinas/genética , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174PPAVP179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.
Asunto(s)
Virus del Dengue/química , Dengue/virología , Quercetina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Virus del Dengue/efectos de los fármacos , Virus del Dengue/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , ARN/metabolismo , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Proteínas no Estructurales Virales/metabolismo , Difracción de Rayos XRESUMEN
An ensemble-modeling scheme incorporating coarse-grained simulations with experimental small-angle X-ray scattering (SAXS) data is applied to dengue virus 2 (DENV2) nonstructural protein 5 (NS5). NS5 serves a key role in viral replication through its two domains that are connected by a 10-residue polypeptide segment. A set of representative structures is generated from a simulated structure pool using SAXS data fitting by the non-negativity least squares (NNLS) or standard ensemble optimization method (EOM) based on a genetic algorithm (GA). It is found that a proper low-energy threshold of the structure pool is necessary to produce a conformational ensemble of two representative structures by both NNLS and GA that agrees well with the experimental SAXS profile. The stability of the constructed ensemble is validated also by molecular dynamics simulations with an all-atom force field. The constructed ensemble successfully revealed the domain-domain orientation and domain-contacting interface of DENV2 NS5. Using experimental data fitting and additional investigations with synthesized data, it is found that energy restraint on the conformational pool is necessary to avoid overinterpretation of experimental data by spurious conformational representations.
Asunto(s)
Virus del Dengue/metabolismo , Proteínas no Estructurales Virales/química , Modelos Químicos , Simulación de Dinámica Molecular , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Zika virus (ZIKV) has emerged as a pathogen of major health concern. The virus relies on its non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA and the nonstructural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. In this study we present for the first time insights into the overall structure of the entire French Polynesia ZIKV NS3 in solution. The protein is elongated and flexible in solution. Solution studies of the individual protease- and helicase domains show the compactness of the two monomeric enzymes as well as the contribution of the 10-residues linker region to the flexibility of the entire NS3. We show also the solution X-ray scattering data of the French Polynesia ZIKV NS5, which is dimeric in solution and switches to oligomers in a concentration-dependent manner. The solution shapes of the MTase and RdRp domains are described. The dimer arrangement of ZIKV NS5 is discussed in terms of its importance for MTase-RdRp communication and concerted interaction with its flexible and monomeric counterpart NS3 during viral replication and capping. The comparison of ZIKV NS3 and -NS5 solution data with the related DENV nonstructural proteins shed light into the similarities and diversities of these classes of enzymes. Finally, the effect of ATPase inhibitors to the enzymatic active ZIKV NS3 and the individual helicase are provided.
Asunto(s)
Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Virus Zika/química , Antioxidantes/farmacología , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Péptido Hidrolasas/química , Conformación Proteica , Dominios Proteicos , Quercetina/farmacología , ARN Helicasas/química , Resveratrol , Dispersión del Ángulo Pequeño , Serina Endopeptidasas/química , Soluciones , Estilbenos/farmacología , Difracción de Rayos X , Virus Zika/enzimologíaRESUMEN
Dengue virus (DENV) nonstructural protein 5 (NS5) consists of a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. The cross-talk between these domains occurs via a ten-residue linker. Recent solution studies of DENV NS5 from all four serotypes (DENV-1 to DENV-4) showed that NS5 adopts multiple conformations owing to its flexible linker and that DENV-4 NS5 is more compact and less flexible compared with NS5 from DENV-1 to DENV-3 [Saw et al. (2015), Acta Cryst. D71, 2309-2327]. Here, using a variety of single, double, triple and quadruple mutants of DENV-4 NS5 combined with solution X-ray scattering studies, insight into the critical residues responsible for the differential flexibility of DENV-4 NS5 is presented. The DENV-4 NS5 mutants K271T and S266N/T267A as well as the deletion mutant ΔS266T267 showed enlarged dimensions and flexibility similar to those of DENV-3 NS5. The data indicate that the residues Lys271, Ser266 and Thr267 are important for the compactness of DENV-4 NS5 and therefore may be critical for the regulation of virus replication. Furthermore, quantitative characterization of the flexibility of these DENV-4 NS5 linker mutants using the ensemble-optimization method revealed that these mutants possess a similar conformational distribution to DENV-3 NS5, confirming that these residues in the linker region cause the higher compactness of DENV-4 NS5.
Asunto(s)
Virus del Dengue/química , Dengue/virología , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Virus del Dengue/genética , Humanos , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Dispersión del Ángulo Pequeño , Proteínas no Estructurales Virales/genética , Difracción de Rayos XRESUMEN
Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase-RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.