RESUMO
Tuberculosis is caused by Mycobacterium tuberculosis (Mtb) and is a deadly disease resulting in the deaths of approximately 1.5 million people with 10 million infections reported in 2018. Recently, a key condensation step in the synthesis of mycolic acids was shown to require ß-ketoacyl-ACP synthase (KasA). A crystal structure of KasA with the small molecule DG167 was recently described, which provided a starting point for using computational structure-based approaches to identify additional molecules binding to this protein. We now describe structure-based pharmacophores, docking and machine learning studies with Assay Central as a computational tool for the identification of small molecules targeting KasA. We then tested these compounds using nanoscale differential scanning fluorimetry and microscale thermophoresis. Of note, we identified several molecules including the Food and Drug Administration (FDA)-approved drugs sildenafil and flubendazole with K d values between 30-40 µM. This may provide additional starting points for further optimization.
RESUMO
Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Feromônios/metabolismo , Streptococcus thermophilus/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Feromônios/química , Streptococcus/genética , Streptococcus/metabolismo , Streptococcus thermophilus/química , Streptococcus thermophilus/genética , Transativadores/genéticaRESUMO
Published Mycobacterium tuberculosis ß-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Animais , Antituberculosos/química , Antituberculosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Modelos Moleculares , Mycobacterium tuberculosis/enzimologiaRESUMO
We report GSK3011724A (DG167) as a binary inhibitor of ß-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , Antituberculosos/farmacologia , Sinergismo Farmacológico , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Animais , Antituberculosos/farmacocinética , Linhagem Celular , Cristalografia , Descoberta de Drogas , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Tuberculose/tratamento farmacológicoRESUMO
Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2Sd). Here, we compared two Rgg2Sd(C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes (rgg2Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp*, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd(C45S) crystal structures. The Rgg2Sp* mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp* mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/química , Modelos Moleculares , Mutação Puntual , Streptococcus pyogenes/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Ciclosporina/farmacologia , Dimerização , Farmacorresistência Bacteriana , Cinética , Mutagênese Sítio-Dirigida , Feromônios/química , Feromônios/metabolismo , Feromônios/farmacologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Streptococcus pyogenes/efeitos dos fármacos , Transativadores/antagonistas & inibidores , Transativadores/química , Transativadores/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Feromônios/genética , Receptores de Feromônios/genética , Streptococcus/genética , Bacillus/classificação , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Feromônios/metabolismo , Filogenia , Percepção de Quorum/genética , Receptores de Feromônios/metabolismo , Transdução de Sinais , Streptococcus/classificação , Streptococcus/metabolismo , Relação Estrutura-Atividade , Transativadores/genética , Transativadores/metabolismoRESUMO
Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/química , Peptídeo Hidrolases/química , Estrutura Terciária de Proteína , Sítios de Ligação , Cristalografia por Raios X , Vírus da Febre Hemorrágica da Crimeia-Congo/patogenicidade , Febre Hemorrágica da Crimeia/virologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Especificidade por Substrato , Ubiquitina/químicaRESUMO
Staphylococcus aureus is one of the most common nosocomial sources of soft-tissue and skin infections and has more recently become prevalent in the community setting as well. Since the use of penicillins to combat S. aureus infections in the 1940s, the bacterium has been notorious for developing resistances to antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA). With the persistence of MRSA as well as many other drug resistant bacteria and parasites, there is a growing need to focus on new pharmacological targets. Recently, class II fructose 1,6-bisphosphate aldolases (FBAs) have garnered attention to fill this role. Regrettably, scarce biochemical data and no structural data are currently available for the class II FBA found in MRSA (SaFBA). With the recent finding of a flexible active site zinc-binding loop (Z-Loop) in class IIa FBAs and its potential for broad spectrum class II FBA inhibition, the lack of information regarding this feature of class IIb FBAs, such as SaFBA, has been limiting for further Z-loop inhibitor development. Therefore, we elucidated the crystal structure of SaFBA to 2.1 Å allowing for a more direct structural analysis of SaFBA. Furthermore, we determined the KM for one of SaFBA's substrates, fructose 1,6-bisphosphate, as well as performed mode of inhibition studies for an inhibitor that takes advantage of the Z-loop's flexibility. Together the data offers insight into a class IIb FBA from a pervasively drug resistant bacterium and a comparison of Z-loops and other features between the different subtypes of class II FBAs.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as Mycobacterium tuberculosis, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC50 of 10 ± 1 µM for the class II FBA present in M. tuberculosis (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant Staphylococcus aureus. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates.
Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Hidroxiquinolinas/farmacologia , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Animais , Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Cinética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Modelos Moleculares , Coelhos , Alinhamento de SequênciaRESUMO
Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.
Assuntos
Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Fosfato de Di-Hidroxiacetona/metabolismo , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/genética , Ácidos Hidroxâmicos/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.
Assuntos
Citocinas/metabolismo , Modelos Moleculares , Nairovirus/enzimologia , Peptídeo Hidrolases/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nairovirus/metabolismo , Nairovirus/patogenicidade , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Conformação Proteica , Alinhamento de Sequência , Especificidade da Espécie , Especificidade por Substrato , Proteínas Virais/química , Fatores de Virulência/químicaRESUMO
Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. ADSL executes two nonsequential steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide, which occurs in the de novo synthesis of IMP, and the conversion of adenylosuccinate to AMP, which occurs in the de novo synthesis of AMP and also in the purine nucleotide cycle, using the same active site. Mutation of ADSL's arginine 303 to a cysteine is known to lead to ADSL deficiency. Interestingly, unlike other mutations leading to ADSL deficiency, the R303C mutation has been suggested to more significantly affect the enzyme's ability to catalyze the conversion of succinyladenosine monophosphate than that of SAICAR to their respective products. To better understand the causation of disease due to the R303C mutation, as well as to gain insights into why the R303C mutation potentially has a disproportional decrease in activity toward its substrates, the wild type (WT) and the R303C mutant of ADSL were investigated enzymatically and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL's cooperativity. By utilizing this information, a model for the interaction between ADSL and SAICAR is proposed.
Assuntos
Adenilossuccinato Liase/química , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Adenilossuccinato Liase/deficiência , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Transtorno Autístico , Humanos , Mutação de Sentido Incorreto , Ribonucleotídeos/metabolismo , Alinhamento de SequênciaRESUMO
Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.
Assuntos
Mycobacterium tuberculosis/enzimologia , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Triose-Fosfato Isomerase/metabolismoRESUMO
Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(-)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 Å, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU's preference for Ub and Ub-like substrates.