RESUMEN
Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.
RESUMEN
Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.
Asunto(s)
Proteínas Bacterianas , Citotoxinas , Proteínas Citotóxicas Formadoras de Poros , Vibrio cholerae , Factores de Virulencia , Membrana Celular/metabolismo , Citotoxinas/química , Citotoxinas/genética , Vibrio cholerae/patogenicidad , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Factores de Virulencia/química , Factores de Virulencia/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Secuencias de Aminoácidos , Mutación , Ácido Glutámico/química , Ácido Glutámico/genéticaRESUMEN
Methionine and cysteine metabolisms are important for the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The transsulfuration pathway converts methionine to cysteine and represents an important link between antioxidant and methylation metabolism in diverse organisms. Using a combination of biochemistry and cryo-electron microscopy, we characterized the first enzyme of the transsulfuration pathway, cystathionine ß-synthase (MtbCbs) in Mtb. We demonstrated that MtbCbs is a heme-less, pyridoxal-5'-phosphate-containing enzyme, allosterically activated by S-adenosylmethionine (SAM). The atomic model of MtbCbs in its native and SAM-bound conformations revealed a unique mode of SAM-dependent allosteric activation. Further, SAM stabilized MtbCbs by sterically occluding proteasomal degradation, which was crucial for supporting methionine and redox metabolism in Mtb. Genetic deficiency of MtbCbs reduced Mtb survival upon homocysteine overload in vitro, inside macrophages, and in mice coinfected with HIV. Thus, the MtbCbs-SAM axis constitutes an important mechanism of coordinating sulfur metabolism in Mtb.
Asunto(s)
Cistationina betasintasa , Mycobacterium tuberculosis , Animales , Microscopía por Crioelectrón , Cistationina betasintasa/química , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cisteína/metabolismo , Metionina/metabolismo , Ratones , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción , Fosfato de Piridoxal/metabolismo , S-Adenosilmetionina/metabolismo , Azufre/metabolismoRESUMEN
Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (ß-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC.
Asunto(s)
Mycobacterium tuberculosis , Ácidos Micólicos , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Hidroliasas/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismoRESUMEN
Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.
Asunto(s)
Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacología , Hidroliasas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Tioacetazona/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Hidroliasas/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Tioacetazona/síntesis química , Tioacetazona/químicaRESUMEN
Background Mycobacterial FASII pathway is governed by the Protein-Protein Interaction mediated dynamics existent between Acyl Carrier Protein and its partner enzymes. The dehydratase HadAB, involved in the third step of FASII synthesis has remained a key target of drugs like Thiacetazone (TAC) and its consequence on AcpM binding is yet to be deciphered. Owing to the transient nature of these interactions, analysing their implications as a drug target has been exhausting. Methods In this context, we have developed an in vitro method to study the effect of thiocarbamide-containing compounds, TAC and SPA0355 (a thiourea analogue) against mycobacterial HadAB. Additionally, by utilizing crypto-ACP (NBD-tagged Acyl Carrier Protein) as a tool of our choice, we attempted at exploring the effect of TAC and SPA0355 on mycobacterial HadAB. Results SPA0355 behaves at par with TAC and undergoes activation in the presence of monooxygenase EthA thus, bringing about a covalent modification in HadA subunit of HadAB. The crypto-ACP method provides insights into the altered substrate housing capability in HadAB associated with the impediment of its AcpM mediated functionality; an outcome attributed to the repercussions associated with the binding of the aforementioned thiourea compounds. Conclusion This investigation has assisted in unveiling a two-step mechanism undertaken by AcpM for interacting with its corresponding partner protein during acyl chain transfer. General significance This study highlights the alterations brought about by drug binding in the interplay between ACP and HadAB. Additionally, this work for the first time establishes the role of SPA0355 as a promising drug candidate against dehydratase HadAB.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Mycobacterium/enzimología , Tiourea/farmacología , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/química , Hidroliasas/metabolismo , Tiourea/análogos & derivados , Tiourea/químicaRESUMEN
In Mycobacterium tuberculosis, acyl carrier protein (AcpM)-mediated fatty acid synthase type II is integral for the synthesis of mycolic acids. AcpM, designated as an atypical ACP, comprises of a putative 33 amino acid long C-terminal extension which is distinctive in nature. Here, we aimed at devising an 'easy-to-go' method for the generation of crypto-AcpM loaded with a solvatochromic probe 7-Nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to the 4'-phosphopantetheine (Ppant) prosthetic group of AcpM. The crypto-AcpM, coupled with fluorescence spectroscopy and molecular dynamics simulation studies, was employed to explore the elusive dynamics of Ppant arm in AcpM. This investigation establishes the role of the flexible C-terminal extension of AcpM in regulating the prosthetic group sequestration ability by modulating the 'Asp-Ser-Leu' motif.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Coenzima A/química , Mycobacterium tuberculosis/química , Panteteína/análogos & derivados , Secuencias de Aminoácidos , Azoles/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Coenzima A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Nitrobencenos/química , Panteteína/química , Panteteína/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.
Asunto(s)
Disulfuros/metabolismo , Conformación Proteica , Staphylococcus aureus/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Disulfuros/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , NADP/metabolismo , Oxidación-Reducción , Especificidad por Sustrato , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/químicaRESUMEN
Matrix metalloproteinases (MMPs) are well-known proteolytic enzymes in animal systems and play roles in tissue differentiation, growth, and defence. Although a few plant MMPs have been reported, their exact functions in development and growth remain elusive. In this study, we characterized the promoter and coding sequence of OsMMP1, one of the putative MMP genes in rice (Oryza sativa). The OsMMP1 catalytic domain is structurally similar to human MMPs with respect to cofactor orientation as predicted by homology modeling. Bacterially expressed recombinant OsMMP1 showed protease activity with bovine serum albumin and gelatin as substrates. Analyses of transcript accumulation and promoter-reporter gene expression revealed that OsMMP1 is spatio-temporally expressed in vegetative and reproductive parts of plants. The plasma membrane-localized OsMMP1 protease affected plant development upon heterologous expression in tobacco and endogenous gene silencing in rice. Transgenic tobacco plants expressing OsMMP1 showed enhanced deposition of cellulose and callose, leading to impairment of symplastic and apoplastic translocations. Moreover, transgenic tobacco tissues exhibited tolerance to oxidative stress-inducing agent by confining the area of tissue death owing to callose lining. Collectively, these findings demonstrate the involvement of a plant MMP in growth, organ differentiation, and development in relation to cell wall modification.
Asunto(s)
Celulosa/metabolismo , Glucanos/metabolismo , Metaloproteinasa 1 de la Matriz/fisiología , Oryza/enzimología , Oryza/crecimiento & desarrollo , Proteínas de Plantas/fisiología , Dominio Catalítico , Pared Celular/metabolismo , Escherichia coli , Regulación de la Expresión Génica de las Plantas , Metaloproteinasa 1 de la Matriz/química , Metaloproteinasa 1 de la Matriz/genética , Oryza/genética , Estrés Oxidativo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/genética , Nicotiana/crecimiento & desarrolloRESUMEN
Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ß strands stacked between α helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Mapeo Epitopo , Secuencia de Aminoácidos , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/inmunología , Conformación ProteicaRESUMEN
Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work, eight compounds have been designed, synthesized, characterized and evaluated for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of them (12-14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar concentration. In addition, the dual inhibitors show good anti-mycobacterial properties against both planktonic growth and biofilm culture of Mycobacterium species. This study is an important addition to tuberculosis drug discovery because it explores two new enzymes as drug targets and presents their dual inhibitors as good candidates for pre-clinical trials.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Antituberculosos/síntesis química , Antituberculosos/química , Biocatálisis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Hidroliasas/metabolismo , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.
Asunto(s)
Enoil-CoA Hidratasa/ultraestructura , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/ultraestructura , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Simulación por Computador , Cristalización , Dimerización , Enoil-CoA Hidratasa/química , Enoil-CoA Hidratasa/metabolismo , Activación Enzimática , Acido Graso Sintasa Tipo II/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mycobacterium tuberculosis/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of Mycobacterium tuberculosis FabG4 (Rv0242c), a less explored ß-ketoacyl CoA reductase that has immense potential to be the future anti-tuberculosis drug target due to its possible involvement in drug resistance and latent infection. Novel triazole linked polyphenol-aminobenzene hybrids have been synthesized, characterized and evaluated for their inhibitory activity against FabG4. All of them inhibit FabG4 at low micromolar concentrations. In silico docking study has been carried out to explain the experimental findings. A comparative study of these new inhibitors with previously reported gallate counterparts leads to structure-activity relations (SAR) of substituent linked to N-1 of triazole ring.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/química , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimología , Triazoles/química , Oxidorreductasas de Alcohol/metabolismo , Antituberculosos/síntesis química , Antituberculosos/farmacología , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Polifenoles/química , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3â M sodium chloride, 0.1â M Na HEPES pH 8.0 and (ii) 2.5â M sodium chloride, 0.1â M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3â Å resolution and belonged to space group I41, with unit-cell parameters a=b=61.51, c=143.81â Å. Crystals from the second condition diffracted to 3.1â Å resolution and belonged to space group P43212 or P41212, with unit-cell parameters a=b=63.67, c=140.88â Å. Both crystals contained one molecule in the asymmetric unit.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Difracción de Rayos X , Clonación Molecular , Cristalización , Homología de Secuencia de AminoácidoRESUMEN
FabGs, or ß-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of ß-oxoacyl-ACP (acyl-carrier protein) into ß-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce ß-oxoacyl-CoA to ß-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD+-hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.