RESUMO
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.
Assuntos
Acinetobacter baumannii , ATPases Translocadoras de Prótons , Humanos , ATPases Translocadoras de Prótons/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Hidrólise , Microscopia Crioeletrônica , Sequência de Aminoácidos , Bactérias/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
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.
Assuntos
Mycobacterium tuberculosis , Mycobacterium , Tuberculose , Humanos , Micobactérias não Tuberculosas , Hidrólise , Trifosfato de AdenosinaRESUMO
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.
Assuntos
Mycobacterium tuberculosis , ATPases Translocadoras de Prótons , Trifosfato de Adenosina/metabolismo , Aminoácidos/farmacologia , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas Recombinantes/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrólise/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Mutação , Mycobacterium , Mycobacterium smegmatis/genética , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
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.
Assuntos
ATPases Translocadoras de Prótons/metabolismo , Proteínas Recombinantes/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Hidrólise , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , ATPases Translocadoras de Prótons/isolamento & purificação , ATPases Translocadoras de Prótons/ultraestrutura , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
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.
Assuntos
Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Desenho de Fármacos , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Mutação , Mycobacterium/crescimento & desenvolvimento , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular , Simulação por Computador , Metabolismo Energético , Modelos Moleculares , Mycobacterium/enzimologia , Mycobacterium/genética , Conformação Proteica , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genéticaRESUMO
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.
Assuntos
Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dimerização , Humanos , Mutação , Conformação Proteica , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Difração de Raios XRESUMO
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.
Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Estabilidade Proteica , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , SoluçõesRESUMO
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.
Assuntos
Vírus da Dengue/química , Dengue/virologia , Quercetina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Aminoácidos , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , RNA/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Proteínas não Estruturais Virais/metabolismo , Difração de Raios XRESUMO
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.
Assuntos
Vírus da Dengue/metabolismo , Proteínas não Estruturais Virais/química , Modelos Químicos , Simulação de Dinâmica Molecular , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
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.
Assuntos
Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Zika virus/química , Antioxidantes/farmacologia , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Peptídeo Hidrolases/química , Conformação Proteica , Domínios Proteicos , Quercetina/farmacologia , RNA Helicases/química , Resveratrol , Espalhamento a Baixo Ângulo , Serina Endopeptidases/química , Soluções , Estilbenos/farmacologia , Difração de Raios X , Zika virus/enzimologiaRESUMO
Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.