RESUMO
Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Sítios de Ligação , Marcação por Isótopo , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2'-phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat /KM ) of NADP(+) at least an order of magnitude lower than NAD(+) . Substitution of Glu42 with Ala improved specificity toward NADP(+) by 10-fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD(+) -OH(-) ) adduct contraposing previously reported adducts. The OH(-) of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2'-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD(+) -OH(-) adduct in proton relay instead of hydride transfer as noted for previous adducts.
Assuntos
Proteínas de Bactérias/química , Ácidos e Sais Biliares/biossíntese , Clostridium/enzimologia , Hidroxiesteroide Desidrogenases/química , Apoenzimas/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , NAD/químicaRESUMO
Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.
Assuntos
Amidas/química , Cromatografia de Fase Reversa/métodos , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Temperatura , Sequência de Aminoácidos , Soluções Tampão , Humanos , Dados de Sequência Molecular , Peptídeos/química , Fatores de TempoRESUMO
Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.
Assuntos
Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/química , Glutationa Transferase/química , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/citologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Cristalografia por Raios X , Dimerização , Glutationa Transferase/classificação , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de ProteínaRESUMO
RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , Metiltransferases/química , RNA Ribossômico/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Metiltransferases/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.
Assuntos
Proteínas de Bactérias/química , Estrutura Terciária de Proteína , Thermotoga maritima/enzimologia , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Infecções Bacterianas/epidemiologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Nucleotídeos de Desoxiuracil/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Dobramento de ProteínaAssuntos
Bacillus subtilis/enzimologia , Organofosfatos/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/fisiologia , Tioglicosídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Hidrólise , Dados de Sequência Molecular , Organofosfatos/química , Tioglicosídeos/químicaAssuntos
Proteínas de Ligação a DNA/química , Thermotoga maritima/metabolismo , Sequência de Aminoácidos , Anisotropia , Cristalografia por Raios X , DNA de Cadeia Simples , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Fatores de Transcrição/química , Difração de Raios XAssuntos
Fosfatase Ácida/química , Clostridium acetobutylicum/enzimologia , Fosfatase Ácida/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Clostridium acetobutylicum/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de ProteínaAssuntos
Dioxigenases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Bases de Dados de Proteínas , Dioxigenases/genética , Dioxigenases/metabolismo , Metais/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaAssuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ciclofilinas/química , Ciclofilinas/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase/classificação , Polietilenoglicóis , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaAssuntos
Proteínas de Bactérias/química , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Thermotoga maritima/química , Proteína ran de Ligação ao GTP/químicaRESUMO
Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.
Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Glicerofosfatos/metabolismo , Imidazóis/farmacologia , Modelos BiológicosRESUMO
Genome-wide gene essentiality data sets are becoming available for Escherichia coli, but these data sets have yet to be analyzed in the context of a genome scale model. Here, we present an integrative model-driven analysis of the Keio E. coli mutant collection screened in this study on glycerol-supplemented minimal medium. Out of 3,888 single-deletion mutants tested, 119 mutants were unable to grow on glycerol minimal medium. These conditionally essential genes were then evaluated using a genome scale metabolic and transcriptional-regulatory model of E. coli, and it was found that the model made the correct prediction in approximately 91% of the cases. The discrepancies between model predictions and experimental results were analyzed in detail to indicate where model improvements could be made or where the current literature lacks an explanation for the observed phenotypes. The identified set of essential genes and their model-based analysis indicates that our current understanding of the roles these essential genes play is relatively clear and complete. Furthermore, by analyzing the data set in terms of metabolic subsystems across multiple genomes, we can project which metabolic pathways are likely to play equally important roles in other organisms. Overall, this work establishes a paradigm that will drive model enhancement while simultaneously generating hypotheses that will ultimately lead to a better understanding of the organism.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Simulação por Computador , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Glicerol , Modelos Biológicos , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
A single base (U1939) within E. coli 23S ribosomal RNA is methylated by its dedicated enzyme, RumA. The structure of RumA/RNA/S-adenosylhomocysteine uncovers the mechanism for achieving unique selectivity. The single-stranded substrate is "refolded" on the enzyme into a compact conformation with six key intra-RNA interactions. The RNA substrate contributes directly to catalysis. In addition to the target base, a second base is "flipped out" from the core loop to stack against the adenine of the cofactor S-adenosylhomocysteine. Nucleotides in permuted sequence order are stacked into the site vacated by the everted target U1939 and compensate for the energetic penalty of base eversion. The 3' hairpin segment of the RNA binds distal to the active site and provides binding energy that contributes to enhanced catalytic efficiency. Active collaboration of RNA in catalysis leads us to conclude that RumA and its substrate RNA may reflect features from the earliest RNA-protein era.