RESUMEN
Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , Lepra/tratamiento farmacológico , Mycobacterium leprae/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/ultraestructura , Clofazimina/farmacología , Dapsona/farmacología , Proteínas de Choque Térmico/ultraestructura , Interacciones Huésped-Patógeno/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Leprostáticos/química , Leprostáticos/farmacología , Lepra/genética , Lepra/inmunología , Lepra/microbiología , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mycobacterium leprae/patogenicidad , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Rifampin/farmacologíaRESUMEN
Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.
Asunto(s)
Antibacterianos/química , Desarrollo de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/enzimología , ARNt Metiltransferasas/antagonistas & inhibidores , ARNt Metiltransferasas/metabolismo , Antibacterianos/farmacología , Cristalografía por Rayos X/métodos , Humanos , Estructura Secundaria de ProteínaAsunto(s)
Pueblo Asiatico/genética , Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Glicina/genética , Clorhidrato de Olopatadina/administración & dosificación , Vitamina E/administración & dosificación , Administración Oral , Administración Tópica , Sustitución de Aminoácidos/genética , Antiinflamatorios no Esteroideos/administración & dosificación , Niño , Colágeno Tipo VII/química , Epidermólisis Ampollosa Distrófica/diagnóstico , Epidermólisis Ampollosa Distrófica/tratamiento farmacológico , Glicina/química , Humanos , Masculino , Estructura Secundaria de Proteína , Resultado del TratamientoRESUMEN
Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded ß-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A ß-hairpin inserted into the first ß-meander extends across the bottom of the structure towards the C-terminal edge of the second ß-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the ß-tent fold for the striking arrangement of its constituent ß-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.
Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Talidomida/química , Talidomida/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/química , Drosophila , Proteínas de Drosophila/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Ubiquitina-Proteína LigasasRESUMEN
The life-threatening infections caused by Mycobacterium leprae (Mle) remain a major challenge in developing countries as well as globe and there is a need to design potent anti-leprosy drugs. In our previous studies, ATP-dependent Mle-MurE ligase involved in biosynthesis of peptidoglycan was identified as one of the common drug targets, homology modeled and reported. In this work in silico site directed mutagenesis study was carried out on the homology modeled Mle-MurE ligase. This predicted the amino acids essential for stability. In addition, the distribution of these residues in different secondary structures and in active sites was analyzed. Finally, the role of the conserved residues in stability and function was analyzed. The availability of Mle-MurE ligase built model together with insights gained from stability studies and docking studies will promote the rational design of potent and selective Mle-MurE ligase inhibitors as anti-leprosy therapeutics.
Asunto(s)
Aminoácidos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mycobacterium leprae/metabolismo , Péptido Sintasas/química , Péptido Sintasas/genética , Adenosina Trifosfato/química , Catálisis , Simulación por Computador , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Humanos , Lepra/tratamiento farmacológico , Ligandos , Mutagénesis Sitio-Dirigida , Peptidoglicano/química , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two ß-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel ß-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the ß-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹5N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.
Asunto(s)
Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Antituberculosos/farmacología , Azitromicina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas Fúngicas/genética , Mycobacterium avium/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Área Bajo la Curva , Secuencia Conservada , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mycobacterium avium/efectos de los fármacos , Mycobacterium avium/metabolismo , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Alineación de Secuencia , Factores de TiempoRESUMEN
Leprosy is an infectious disease caused by Mycobacterium leprae. The increasing drug and multi-drug resistance of M. leprae enforce the importance of finding new drug targets. Mycobacterium has unusually impermeable cell wall that contributes to considerable resistance to many drugs. Peptidoglycan is an important component of the cell wall of M. leprae. UDP-N-acetylmuramoyl-glycyl-D-glutamate-2, 6-diaminopimelate ligase (MurE) plays a crucial role in the peptidoglycan biosynthesis and hence it could be considered as a potential drug target for leprosy. Structure of this enzyme for M. leprae has not yet been elucidated. We modeled the three-dimensional structure of MurE from M. leprae using comparative modeling methods based on the X-ray crystal structure of MurE from E. coli and validated. The 3D-structure of M. leprae MurE enzyme was docked with its substrates meso-diaminopimelic acid (A2pm) and UDP-N-acetyl muramoyl-glycyl-D- glutamate (UMGG) and its product UDP-N-acetyl muramoyl-glycyl-D-glu-meso-A(2)pm (UTP) and also with ATP. The docked complexes reveal the amino acids responsible for binding the substrates. Superposition of these complex structures suggests that carboxylic acid group of UMGG is positioned in proximity to γ-phosphate of the ATP to facilitate the formation of acylphosphate intermediate. The orientation of an amino group of A(2)pm facilitates the nucleophilic attack to form the product. Overall, the proposed model together with its binding features gained from docking studies could help to design a truly selective ligand inhibitor specific to MurE for the treatment of leprosy.
Asunto(s)
Modelos Moleculares , Mycobacterium leprae/enzimología , Péptido Sintasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Escherichia coli/enzimología , Modelos Químicos , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.
Asunto(s)
Antifúngicos/metabolismo , Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Econazol/metabolismo , Lepra/tratamiento farmacológico , Mycobacterium leprae , Antifúngicos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Econazol/farmacología , Hemo/metabolismo , Histidina/metabolismo , Humanos , Hierro/metabolismo , Cinética , Lepra/microbiología , Modelos Moleculares , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/enzimología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/enzimología , Porfirinas/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Lepra/microbiología , Mycobacterium leprae/enzimología , Péptido Sintasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Diseño de Fármacos , Ácido Glutámico/química , Glicina/química , Histidina/química , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Homología Estructural de Proteína , TermodinámicaRESUMEN
Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.
Asunto(s)
Mycobacterium leprae/enzimología , Mycobacterium tuberculosis/enzimología , Rec A Recombinasas/química , Rec A Recombinasas/fisiología , Homología Estructural de Proteína , Secuencia de Aminoácidos , Composición de Base , Sitios de Unión/genética , Clonación Molecular , ADN de Cadena Simple/metabolismo , Datos de Secuencia Molecular , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Unión Proteica , Estructura Secundaria de Proteína , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Spectroscopic and thermodynamic properties were determined for Debaryomyces hansenii UFV-1 extracellular and intracellular alpha-galactosidases. alpha-Galactosidases showed similar secondary structure compositions (alpha-helix, beta-sheet parallel and beta-turn). Effects of pH and temperature on the structure of alpha-galactosidases were investigated using circular dichroism spectroscopy. It was more pronounced at low pH. Microcalorimetry was employed for the determination of thermodynamic parameters. Immediate thermal denaturation reversibility was not observed for alpha-galactosidases; it occurred as a thermodynamically driven process. Extracellular alpha-galactosidase, at pH 5.5, showed lower T(m) when compared to the intracellular enzyme. The CD and DSC data suggest that D. hansenii alpha-galactosidases have different behaviors although they possess some similar secondary structures.
Asunto(s)
Dicroismo Circular , Debaryomyces/enzimología , alfa-Galactosidasa/química , Rastreo Diferencial de Calorimetría , Espacio Extracelular/enzimología , Concentración de Iones de Hidrógeno , Desnaturalización Proteica , Estructura Secundaria de Proteína , Temperatura , Termodinámica , Temperatura de Transición , alfa-Galactosidasa/metabolismoRESUMEN
Mycobacterium leprae protein ML2640c belongs to a large family of conserved hypothetical proteins predominantly found in mycobacteria, some of them predicted as putative S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTase). As part of a Structural Genomics initiative on conserved hypothetical proteins in pathogenic mycobacteria, we have determined the structure of ML2640c in two distinct crystal forms. As expected, ML2640c has a typical MTase core domain and binds the methyl donor substrate AdoMet in a manner consistent with other known members of this structural family. The putative acceptor substrate-binding site of ML2640c is a large internal cavity, mostly lined by aromatic and aliphatic side-chain residues, suggesting that a lipid-like molecule might be targeted for catalysis. A flap segment (residues 222-256), which isolates the binding site from the bulk solvent and is highly mobile in the crystal structures, could serve as a gateway to allow substrate entry and product release. The multiple sequence alignment of ML2640c-like proteins revealed that the central alpha/beta core and the AdoMet-binding site are very well conserved within the family. However, the amino acid positions defining the binding site for the acceptor substrate display a higher variability, suggestive of distinct acceptor substrate specificities. The ML2640c crystal structures offer the first structural glimpses at this important family of mycobacterial proteins and lend strong support to their functional assignment as AdoMet-dependent methyltransferases.
Asunto(s)
Metiltransferasas/química , Mycobacteriaceae/enzimología , Mycobacterium leprae/enzimología , S-Adenosilmetionina/química , Secuencia de Aminoácidos , Sitios de Unión , Biología Computacional/métodos , Cristalografía por Rayos X , Bases de Datos de Proteínas , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacteriaceae/genética , Mycobacterium leprae/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , Especificidad por SustratoRESUMEN
Co-chaperonins from diverse organisms exhibit mobile loops which fold into a beta hairpin conformation upon binding to the chaperonin. GroES, Gp31, and human Hsp10 mobile loops exhibit a preference for the beta hairpin conformation in the free co-chaperonins, and the conformational dynamics of the human Hsp10 mobile loop appear to be restricted by nascent hairpin formation. Backbone conformational entropy must weigh against binding of co-chaperonins to chaperonins, and thus the conformational preferences of the loops may strongly influence chaperonin-binding affinity. Indeed, subtle mutations in the loops change GroEL-binding affinity and cause defects in chaperonin function, and these defects can be suppressed by mutations in GroEL which compensate for the changes in affinity. The fact that high-affinity co-chaperonin binding impairs chaperonin function has implications for the mechanism of chaperonin-assisted protein folding.
Asunto(s)
Chaperoninas/química , Chaperoninas/metabolismo , Secuencia de Aminoácidos , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Gráficos por Computador , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium leprae/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
SETTING: Molecular Research Laboratory, Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital. OBJECTIVE: Characterize Mycobacterium tuberculosis homologue of the Streptomyces coelicolor, sporulation specific, whiB regulatory gene. DESIGN: The M. tuberculosis whiB3 gene was isolated by enriched cloning of a 2.8 kb BamHl fragment to which the S. coelicolor whiB gene hybridized. Expression of the gene was analysed by S1 nuclease analysis and promoter studies. RESULTS: An open reading frame within the 2.8 kb BamHl fragment was identified as the M. tuberculosis whiB3 gene, one of four whiB homologues in the M. tuberculosis genome. The deduced amino acid sequence has a 92% identity with a M. leprae protein, and 32% identity with the S. coelicolor WhiB protein. S1 nuclease analysis showed that the M. tuberculosis whiB3 gene is constitutively expressed by the cells in liquid culture. Primer extension analysis revealed three transcriptional start sites. Expression from the three potential promoters is growth phase-dependent. CONCLUSION: The M. tuberculosis whiB3 gene is expressed throughout growth, but expression from the individual promoters is growth phase dependent.
Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Streptomyces/genética , Factores de Transcripción/genética , Proteínas Bacterianas/química , Southern Blotting , Humanos , Mycobacterium/genética , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , ARN Bacteriano/genética , ARN Mensajero/genética , Factores de Transcripción/químicaRESUMEN
Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer-tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.
Asunto(s)
ADN Helicasas , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Conformación Proteica , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X/métodos , Escherichia coli , Proteínas de Escherichia coli , Sustancias Macromoleculares , Modelos Moleculares , Mycobacterium leprae , Pliegue de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.
Asunto(s)
Estructura Secundaria de Proteína , Virus ARN/enzimología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Virus ARN/clasificación , Virus ARN/fisiología , Alineación de Secuencia , Replicación ViralRESUMEN
Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins. The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms. The architecture of the Ml-cpn10 heptamer resembles a dome with an oculus in its roof. The inner surface of the dome is hydrophilic and highly charged. A flexible region, known to interact with cpn60, extends from the lower rim of the dome. With the structure of a cpn10 heptamer now revealed and the structure of the E. coli GroEL previously known, models of cpn10:cpn60 and GroEL:GroES complexes are proposed.
Asunto(s)
Chaperonina 10/química , Mycobacterium leprae/química , Conformación Proteica , Secuencia de Aminoácidos , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
Arylsulphatase activity was identified in cultures of the marine bacterium Alteromonas carrageenovora, using methylumbelliferyl sulphate as substrate. In contrast with most other microbial arylsulphatases, arylsulphatase production in A. carrageenovora was not repressed by sulphate. The structural gene of arylsulphatase (atsA) was cloned and sequenced. An ORF of 984 bp was found, specifying a primary translation product of 328 amino acids with a molecular mass of 35797 Da. Arylsulphatase was partially purified from cell extracts of both A. carrageenovora and recombinant Escherichia coli. Both the recombinant and native enzymes exhibited a pI of 5.5, a Michaelis constant for methylumbelliferyl sulphate of 68 microM, and a molecular mass of approximately 35,000 Da in SDS-PAGE analysis. Secondary structure comparisons using hydrophobic cluster analysis suggest functional analogies between the arylsulphatase of A. carrageenovora, that of Mycobacterium leprae and a 33.5 kDa protein from Porphyromonas gingivalis. It is speculated that these proteins are all glycosulphohydrolases, involved with desulphatation of sulphated polysaccharides.