RESUMO
Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and can act as an important substrate in numerous biological processes. As a result, nature has evolved multiple biosynthetic pathways to meet this high chemical demand. In Saccharomyces cerevisiae, the NAD salvage pathway relies on the activity of nicotinic acid phosphoribosyltransferase (NAPRTase), a member of the phosphoribosyltransferase (PRTase) superfamily. Here, we report the structure of a eukaryotic (yeast) NAPRTase at 1.75 A resolution (locus name: YOR209C, gene name: NPT1). The structure reveals a two-domain fold that resembles the architecture of quinolinic acid phosphoribosyltransferases (QAPRTases), but with completely different dispositions that provide evidence for structural heterogeneity among the Type II PRTases. The identification of a third domain in NAPRTases provides a structural basis and possible mechanism for the functional modulation of this family of enzymes by ATP.
Assuntos
Pentosiltransferases/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , NAD/biossíntese , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
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
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 ProteínaRESUMO
Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.
Assuntos
Proteínas de Bactérias/análise , Proteoma/análise , Thermotoga maritima/química , Sequência de Aminoácidos , Aminoácidos/química , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Ponto Isoelétrico , Fases de Leitura Aberta , Conformação Proteica , Sinais Direcionadores de Proteínas , Thermotoga maritima/genéticaRESUMO
The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Subunidades Proteicas/química , Animais , Bovinos , AMP Cíclico/química , AMP Cíclico/farmacologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico , Evolução Molecular , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Soluções , Difração de Raios XRESUMO
Recently, the structures of two proteins belonging to the archease family, TM1083 from Thermotoga maritima and MTH1598 from Methanobacterium thermoautotrophicum, have been solved independently by two Protein Structure Initiative structural genomics pilot centers using X-ray crystallography and NMR, respectively. The archease protein family is a good example of one of the paradoxes of structural genomics: Approximately one third of protein structures produced by structural genomics centers have no known function and are still annotated as "hypothetical proteins" in the Protein Data Bank. In the case of archeases, despite the existence of two protein structures and abundant sequence information, there is still no function assigned to this protein family. Here, our group predicts, based on structural similarity, sequence conservation, and gene context analyses, that members of this protein family might function as chaperones or modulators of proteins involved in DNA/RNA processing. The conservation of genomic context for this protein family is constant from Archaea and Bacteria to humans, and suggests that unannotated open reading frames contiguous to them could be novel RNA/DNA binding proteins.
Assuntos
Genômica , Proteínas/química , Proteínas/metabolismo , Homologia Estrutural de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Archaea/química , Archaea/genética , Bactérias/química , Bactérias/genética , Sítios de Ligação , Biologia Computacional , Sequência Conservada , Perfilação da Expressão Gênica , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta/genética , Estrutura Terciária de Proteína , Proteínas/genética , Proteômica , Alinhamento de Sequência , Homologia de Sequência , Relação Estrutura-AtividadeRESUMO
Sequence analysis of individual targets is an important step in annotation and validation. As a test case, we investigated human breast cancer associated gene 3 (BCA3) with LION Target Engine and with other bioinformatics tools. LION Target Engine confirmed that the BCA3 gene is located on 11p15.4 and that the two most likely splice variants (lacking exon 3 and exons 3 and 5, respectively) exist. Based on our manual curation of sequence data, it is proposed that an additional variant (missing only exon 5) published in a public sequence repository, is a prediction artifact. A significant number of new orthologs were also identified, and these were the basis for a high-quality protein secondary structure prediction. Moreover, our research confirmed several distinct functional domains as described in earlier reports. Sequence conservation from multiple sequence alignments, splice variant identification, secondary structure predictions, and predicted phosphorylation sites suggest that the removal of interaction sites through alternative splicing might play a modulatory role in BCA3. This in silico approach shows the depth and relevance of an analysis that can be accomplished by including a variety of publicly available tools with an integrated and customizable life science informatics platform.