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Dengue virus (DENV) infection continues to be a public health challenge, lacking a specific cure. Vaccination remains the primary strategy against dengue; however, existing live-attenuated vaccines display variable efficacy across four serotypes, influenced by host serostatus and age, and predominantly inducing humoral responses. To address this limitation, this study investigates a multiepitope-based immunogen designed to induce robust cellular immunity across all DENV serotypes. The chimeric immunogen integrates H-2d specific MHC-I binding T-cell epitopes derived from conserved domains within the DENV envelope protein. Immuno-informatics analyses supported its stability, non-allergenic nature, and strong MHC-I binding affinity as an antigen. To assess the immunogenicity of the multiepitope, it was expressed in murine bone-marrow-derived dendritic cells (BMDCs) that were used to prime mice. In this experimental model, simultaneous exposure to T-cell epitopes from all four DENV serotypes initiated distinct IFNγ-CD8 T-cell responses for different serotypes. These results supported the potential of the multiepitope construct as a vaccine candidate. While the optimization of the immunogen design remains a continuous pursuit, this proof-of-concept study provides a starting point for evaluating its protective efficacy against dengue infection in vivo. Moreover, our results support the development of a multiepitope vaccine that could trigger a pan-serotype anti-dengue CD8 response.
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The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies. Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.
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Proteolisis , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Animales , Humanos , Camélidos del Nuevo Mundo , Procolágeno/metabolismo , Procolágeno/química , Dominios Proteicos , Modelos Moleculares , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Cristalografía por Rayos XRESUMEN
Red luminophores displaying large Stokes shift and high-quantum yields are obtained when gold salts are reacted with proteins under strongly alkaline conditions. Although bovine serum albumin (BSA) has mainly been used as a protein template, other attempts to prepare red luminophores have been proposed using other proteins. Here, we report on the structural characterization and nonlinear optical properties of insulin-gold conjugates. Such conjugates display strong luminescence at â¼670 nm with quantum yields that reach 5.4%. They also display long luminescence lifetimes allowing efficient reactive oxygen species generation, with a quantum yield of 1O2 generation reaching 13%. In addition, they exhibit remarkable nonlinear optical properties and in particular a strong two-photon excited fluorescence (TPEF) cross section in the range of 800-1100 nm. By combining experimental studies and time-dependent density functional theory simulations (TD-DFT), we show the formation of insulin-Au(III) conjugates. The interaction of Au(III) ions with the aromatic rings of tyrosine induces charge transfer-like excitation in the visible range. Experimental investigations, together with molecular dynamics simulations of insulin and calculations of electronic properties in a model system, are performed to explore the origin of optical features and the structure-optical property relationship, leading the way to new concepts for nonlinear optics using protein-Au(III) conjugates.
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This article presents bioconjugates combining nanoparticles (AGuIX) with nanobodies (VHH) targeting Programmed Death-Ligand 1 (PD-L1, A12 VHH) and Cluster of Differentiation 47 (CD47, A4 VHH) for active tumor targeting. AGuIX nanoparticles offer theranostic capabilities and an efficient biodistribution/pharmacokinetic profile (BD/PK), while VHH's reduced size (15 kDa) allows efficient tumor penetration. Site-selective sortagging and click chemistry were compared for bioconjugation. While both methods yielded bioconjugates with similar functionality, click chemistry demonstrated higher yield and could be used for the conjugation of various VHH. The specific targeting of AGuIX@VHH has been demonstrated in both in vitro and ex vivo settings, paving the way for combined targeted immunotherapies, radiotherapy, and cancer imaging.
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Gadolinio , Nanopartículas , Neoplasias , Humanos , Distribución Tisular , Medicina de Precisión , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológicoRESUMEN
Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.
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Proteínas Serina-Treonina Quinasas , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ácidos Teicoicos/metabolismo , Proteínas Bacterianas/metabolismo , División Celular , Proteínas Quinasas/metabolismo , Hidrolasas/metabolismo , Pared Celular/metabolismoRESUMEN
Metazoans establish mutually beneficial interactions with their resident microorganisms. However, our understanding of the microbial cues contributing to host physiology remains elusive. Previously, we identified a bacterial machinery encoded by the dlt operon involved in Drosophila melanogaster's juvenile growth promotion by Lactiplantibacillus plantarum. Here, using crystallography combined with biochemical and cellular approaches, we investigate the physiological role of an uncharacterized protein (DltE) encoded by this operon. We show that lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarumNC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarumNC8 and Drosophila melanogaster as a symbiosis model, we establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. Our results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.
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Fenómenos Biológicos , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ácidos Teicoicos/metabolismo , Señales (Psicología) , Lipopolisacáridos/metabolismoRESUMEN
The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.
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Brucelosis , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Núcleo Celular/metabolismo , Brucella abortus/metabolismo , Nucléolo Celular/metabolismo , Brucelosis/microbiología , Chaperonas Moleculares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX(2) RX(2) R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.
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Adenosina Difosfato/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Polisacáridos Bacterianos/metabolismo , Proteínas Tirosina Quinasas/química , Secuencias de Aminoácidos/genética , Cristalografía por Rayos X , Análisis Mutacional de ADN , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismoRESUMEN
Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.
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Proteínas Bacterianas/química , Proteínas Tirosina Quinasas/química , Staphylococcus aureus/enzimología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.
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Arabidopsis/metabolismo , Cobre/metabolismo , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Enfermedades de las Plantas/microbiología , Especies Reactivas de Oxígeno/metabolismo , Antiinfecciosos Locales/farmacología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Botrytis/genética , Botrytis/metabolismo , Proteínas Portadoras/metabolismo , Defensinas/genética , Dickeya , Dimerización , Gammaproteobacteria/efectos de los fármacos , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas de Unión a Hierro/genética , Enfermedades de las Plantas/genética , Sideróforos/genética , Sideróforos/metabolismoRESUMEN
Genome sequencing projects have revealed that P-loop proteins are highly represented in all organisms and that many of them have no attributed function. They are characterized by a conserved nucleotide-binding domain and carry different activities implicated in many cellular processes. Saccharomyces cerevisiae YFH7 is one of these P-loop proteins of unknown function. In this work we tried to integrate bioinformatics, structure, and enzymology to discover the function of YFH7. Sequence analysis revealed that yeast YFH7 is a yeast-specific protein showing weak similarity with the phosphoribulokinase/uridine kinase/bacterial pantothenate kinase (PRK/URK/PANK) subfamily of P-loop containing kinases. A large insertion of about 100 residues distinguishes YFH7 from other members of the family. The 1.95 A resolution crystal structure of YFH7 solved using the SAD method confirmed that YFH7 has a fold similar to the PRK/URK/PANK family, with the characteristic core, lid, and NMP(bind) domains. An additional alpha/beta domain of novel topology corresponds to the large sequence insertion. Structural and ligand binding analysis combined with enzymatic assays suggest that YFH7 is an ATP-dependent small molecule kinase with new substrate specificity.
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Proteínas Quinasas/química , Proteínas de Saccharomyces cerevisiae/química , Sitios de Unión , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
In response to infection by the pathogen Agrobacterium tumefaciens, plants synthesize several stress amino acids, including gamma-aminobutyric acid (GABA), which modulates the expression of bacterial virulence factors. GABA penetrates into the bacterial cytoplasm via an ABC transporter that is associated with the periplasmic receptor Atu2422. Mature receptor Atu2422 (without its signal peptide) was overexpressed in Escherichia coli, purified and crystallized. A complete data set was collected to 1.35 A resolution at 100 K. The crystals belonged to the monoclinic space group C2 and contained one molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed a closed form of this Venus flytrap (VFT) receptor, suggesting the presence of an endogenous E. coli ligand.
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Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/química , Receptores de GABA/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Receptores de GABA/genética , Receptores de GABA/aislamiento & purificaciónRESUMEN
BACKGROUND: Tissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. One of the compensatory mechanisms to alleviate the sepsis-induced mitochondrial dysfunction could be the increase in oxidative phosphorylation efficiency (ATP/O). We propose to study liver mitochondrial function and oxidative stress and the regulatory mechanism of mitochondrial oxidative phosphorylation efficiency in an animal model of sepsis. METHODS: We induced sepsis in rats by cecal ligation and perforation (CLP). Six, 24, or 36 h following CLP, we measured liver mitochondrial respiration, cytochrome c oxidase activity, and membrane permeability. We determine oxidative phosphorylation efficiency, by measuring ATP synthesis related to oxygen consumption at various exogenous ADP concentrations. Finally, we measured radical oxygen species (ROS) generation by liver mitochondria and mRNA concentrations of UCP2, biogenesis factors, and cytokines at the same end points. RESULTS: CLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit ß content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression. CONCLUSIONS: Despite a compensatory increase in mitochondrial biogenesis factors, liver mitochondrial functions remain altered after CLP. This suggests that the functional compensatory mechanisms reported in the present study (slip at cytochrome c oxidase and biogenesis factors) were not strong enough to increase oxidative phosphorylation efficiency and failed to limit liver mitochondrial ROS over-generation. These data suggest that treatments based on cytochrome c infusion could have a role in mitochondrial dysfunction and/or ROS generation associated with sepsis.
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Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.
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División Celular , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Streptococcus pneumoniae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Modelos Moleculares , N-Acetil Muramoil-L-Alanina Amidasa , Fosforilación , Estructura Terciaria de Proteína , Streptococcus pneumoniae/citologíaRESUMEN
Carbon catabolite repression (CCR) in Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase (HprK/P). This enzyme catalyses the ATP- as well as the pyrophosphate-dependent phosphorylation of Ser-46 in HPr, a phosphocarrier protein of a sugar transport and phosphorylation system. HprK/P also catalyses the pyrophosphate-producing, inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr (P-Ser-HPr). P-Ser-HPr functions as catabolite co-repressor by interacting with the LacI/GalR-type repressor, catabolite control protein A (CcpA), and allowing it to bind to operator sites preceding catabolite-regulated transcription units. HprK/P thus indirectly controls the expression of about 10% of the genes of Gram-positive bacteria. The two antagonistic activities of HprK/P are regulated by intracellular metabolites, which change their concentration in response to the absence or presence of rapidly metabolisable carbon sources (glucose, fructose, etc.) in the growth medium. Biochemical and structural studies revealed that HprK/P exhibits no similarity to eukaryotic protein kinases and that it contains a Walker motif A (or P-loop) as nucleotide binding site. Interestingly, HprK/P has a structural fold resembling that in kinases phosphorylating certain low molecular weight substrates such as nucleosides, nucleotides or oxaloacetate. The structures of the complexes of HprK/P with HPr and P-Ser-HPr have also been determined, which allowed proposing a detailed mechanism for the kinase and phosphorylase functions of HprK/P.
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Bacterias Grampositivas/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genes Bacterianos , Bacterias Gramnegativas/enzimología , Modelos Moleculares , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismoRESUMEN
T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.
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Bacteriófago T4/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/genética , Mutación , Uridina Difosfato/química , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Glucosa/química , Iones , Manganeso/química , Metales/química , Modelos Moleculares , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Uridina Difosfato Glucosa/químicaRESUMEN
A particular class of tyrosine-kinases sharing no structural similarity with eukaryotic tyrosine-kinases has been evidenced in a large array of bacterial species. These bacterial tyrosine-kinases are able to autophosphorylate on a C-terminal tyrosine-rich motif. Their autophosphorylation has been shown to play a crucial role in the biosynthesis or export of capsular polysaccharide. The analysis of the first crystal structure of the staphylococcal tyrosine kinase CapB2 associated with the activating domain of the transmembrane modulator CapA1 had brought conclusive explanation for both the autophosphorylation and activation processes. In order to explain why CapA1 activates CapB2 more efficiently than its cognate transmembrane modulator CapA2, we solved the crystal structure of CapA2B2 and compared it with the previously published structure of CapA1B2. This structural analysis did not provide the expected clues about the activation discrepancy observed between the two modulators. Staphylococcus aureus also encodes for a CapB2 homologue named CapB1 displaying more than 70% sequence similarity and being surprisingly nearly unable to autophosphorylate. We solved the crystal structure of CapA1B1 and carefully compare it with the structure of CapA1B2. The active sites of both proteins are highly conserved and the biochemical characterization of mutant proteins engineered to test the importance of small structural discrepancies identified between the two structures did not explain the inactivity of CapB1. We thus tested if CapB1 could phosphorylate other protein substrates or hydrolyze ATP. However, no activity could be detected in our in vitro assays. Taken together, these data question about the biological role of the homologous protein pairs CapA1/CapB1 and CapA2/CapB2 and we discuss about several possible interpretations.
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Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Proteínas Tirosina Quinasas/química , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Staphylococcus aureus/enzimología , Homología Estructural de ProteínaAsunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Fosfoproteínas/química , Serina/química , Cristalografía por Rayos X , Dimerización , Escherichia coli/metabolismo , Modelos Moleculares , Fosforilación , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteómica/métodosAsunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas Represoras/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fructosadifosfatos/química , Fructosadifosfatos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD(+) degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H(2)O(2) produced during the oxidative process or added exogenously.