RESUMEN
Outer membrane beta-barrels (OMBBs) are the major class of outer membrane proteins from Gram-negative bacteria, mitochondria, and plastids. Their transmembrane domains consist of 8-24 beta-strands forming a closed, barrel-shaped beta-sheet around a central pore. Despite their obvious structural regularity, evidence for an origin by duplication or for a common ancestry has not been found. We use three complementary approaches to show that all OMBBs from Gram-negative bacteria evolved from a single, ancestral beta beta hairpin. First, we link almost all families of known single-chain bacterial OMBBs with each other through transitive profile searches. Second, we identify a clear repeat signature in the sequences of many OMBBs in which the repeating sequence unit coincides with the structural beta beta hairpin repeat. Third, we show that the observed sequence similarity between OMBB hairpins cannot be explained by structural or membrane constraints on their sequences. The third approach addresses a longstanding problem in protein evolution: how to distinguish between a very remotely homologous relationship and the opposing scenario of "sequence convergence." The origin of a diverse group of proteins from a single hairpin module supports the hypothesis that, around the time of transition from the RNA to the protein world, proteins arose by amplification and recombination of short peptide modules that had previously evolved as cofactors of RNAs.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Biología Computacional/métodos , Evolución Molecular , Bacterias Gramnegativas/química , Estructura Secundaria de Proteína , Proteínas de la Membrana Bacteriana Externa/genética , Análisis por Conglomerados , Bases de Datos de Proteínas , Bacterias Gramnegativas/genética , Cadenas de Markov , Modelos Biológicos , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mapeo Cromosómico , Deltaproteobacteria/genética , Genoma Bacteriano , Sedimentos Geológicos/microbiología , Regiones Árticas , Proteínas Bacterianas/genética , Composición de Base , Secuencia de Bases , Congelación , Orden Génico , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADNRESUMEN
Proteasomes are large, multisubunit proteases that are found, in one form or another, in all domains of life and play a critical role in intracellular protein degradation. Although they have substantial structural similarity, the proteasomes of bacteria, archaea, and eukaryotes show many differences in architecture and subunit composition. This article discusses possible paths by which proteasomes may have evolved from simple precursors to the highly complicated and diverse complexes observed today.
Asunto(s)
Cisteína Endopeptidasas , Evolución Molecular , Complejos Multienzimáticos , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Compartimento Celular , Cisteína Endopeptidasas/genética , Endopeptidasas/clasificación , Endopeptidasas/genética , Células Eucariotas/enzimología , Células Eucariotas/ultraestructura , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Filogenia , Células Procariotas/enzimología , Células Procariotas/ultraestructura , Complejo de la Endopetidasa ProteasomalRESUMEN
Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Retículo Endoplásmico/metabolismo , Ribonucleoproteínas/fisiología , Animales , Animales Modificados Genéticamente , Southern Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calnexina , Calreticulina , Adhesión Celular , Quimiotaxis , ADN Complementario/metabolismo , Dictyostelium , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Modelos Genéticos , Mutación , Fagocitosis , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Tiempo , Transformación GenéticaRESUMEN
Chaperonesare an essential component of a cell's ability to respond to environmental challenges. Chaperones have been studied primarily in bacteria, but in recent years it has become apparent that some classes of chaperones either are very divergent in bacteria relative to archaea and eukaryotes or are missing entirely. In contrast, a high degree of similarity was found between the chaperonins of archaea and those of the eukaryotic cytosol, which has led to the establishment of archaeal model systems. The archaeon most extensively used for such studies is Thermoplasma acidophilum, which thrives at 59 degrees C and pH 2. Here we review information on its chaperone complement in light of the recently determined genome sequence.
Asunto(s)
Chaperonas Moleculares/química , Thermoplasma/química , Proteínas Arqueales/química , Proteínas Arqueales/genética , Genoma Bacteriano , Chaperonas Moleculares/clasificación , Chaperonas Moleculares/genéticaRESUMEN
This paper presents and discusses evidence suggesting how the diversity of domain folds in existence today might have evolved from peptide ancestors. We apply a structure similarity detection method to detect instances where localized regions of different protein folds contain highly similar sequences and structures. Results of performing an all-on-all comparison of known structures are described and compared with other recently published findings. The numerous instances of local sequence and structure similarities within different protein folds, together with evidence from proteins containing sequence and structure repeats, argues in favor of the evolution of modern single polypeptide domains from ancient short peptide ancestors (antecedent domain segments (ADSs)). In this model, ancient protein structures were formed by self-assembling aggregates of short polypeptides. Subsequently, and perhaps concomitantly with the evolution of higher fidelity DNA replication and repair systems, single polypeptide domains arose from the fusion of ADSs genes. Thus modern protein domains may have a polyphyletic origin.
Asunto(s)
Secuencias de Aminoácidos/genética , Proteínas de Escherichia coli , Evolución Molecular , Péptidos/química , Pliegue de Proteína , Proteínas de Unión al ARN , Secuencia de Aminoácidos/genética , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Biología Computacional/métodos , Grupo Citocromo c/química , Grupo Citocromo c/genética , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Humanos , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfotransferasas/química , Fosfotransferasas/genéticaRESUMEN
We applied a new protocol based on PSI-Blast to predict the structures of fold recognition targets during CASP4. The protocol used a back-validation step to infer biologically significant connections between sequences with PSI-Blast E-values up to 10. If connections were found to proteins of known structure, alignments were generated by using HMMer. The protocol was implemented in a fully automated version (SBauto) and in a version that allowed manual intervention (SBfold). We found that the automated version made 17 predictions for target domains, of which 8 identified the correct fold with an average alignment accuracy of 24% for alignable residues and 43% for equivalent secondary structure elements. The manual version improved predictions somewhat, with 10 of 15 predictions identifying the correct fold with alignment accuracies of 33% for alignable residues and 64% for equivalent secondary structure elements. We describe successes and failures of our approach and discuss future developments of fold recognition.
Asunto(s)
Conformación Proteica , Pliegue de Proteína , Alineación de Secuencia , Ácido Anhídrido Hidrolasas/química , Secuencia de Aminoácidos , Automatización , Proteínas Bacterianas/química , Hidrolasas de Éster Carboxílico/química , Simulación por Computador , Bases de Datos de Proteínas , Geobacillus stearothermophilus , Glicósido Hidrolasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa , Polisacárido Liasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Programas Informáticos , Streptococcus mutans , Factores de Transcripción/químicaRESUMEN
Two-component signal transduction (TCST) systems are the principal means for coordinating responses to environmental changes in bacteria as well as some plants, fungi, protozoa, and archaea. These systems typically consist of a receptor histidine kinase, which reacts to an extracellular signal by phosphorylating a cytoplasmic response regulator, causing a change in cellular behavior. Although several model systems, including sporulation and chemotaxis, have been extensively studied, the evolutionary relationships between specific TCST systems are not well understood, and the ancestry of the signal transduction components is unclear. Phylogenetic trees of TCST components from 14 complete and 6 partial genomes, containing 183 histidine kinases and 220 response regulators, were constructed using distance methods. The trees showed extensive congruence in the positions of 11 recognizable phylogenetic clusters. Eukaryotic sequences were found almost exclusively in one cluster, which also showed the greatest extent of domain variability in its component proteins, and archaeal sequences mainly formed species-specific clusters. Three clusters in different parts of the kinase tree contained proteins with serine-phosphorylating activity. All kinases were found to be monophyletic with respect to other members of their superfamily, such as type II topoisomerases and Hsp90. Structural analysis further revealed significant similarity to the ATP-binding domain of eukaryotic protein kinases. TCST systems are of bacterial origin and radiated into archaea and eukaryotes by lateral gene transfer. Their components show extensive coevolution, suggesting that recombination has not been a major factor in their differentiation. Although histidine kinase activity is prevalent, serine kinases have evolved multiple times independently within this family, accompanied by a loss of the cognate response regulator(s). The structural and functional similarity between TCST kinases and eukaryotic protein kinases raises the possibility of a distant evolutionary relationship.
Asunto(s)
Evolución Molecular , Proteínas Quinasas/genética , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Transferencia de Gen Horizontal , Ligamiento Genético , Histidina Quinasa , Datos de Secuencia Molecular , Fosforilación , Filogenia , Estructura Terciaria de Proteína , Homología de SecuenciaRESUMEN
Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.
Asunto(s)
Genoma Arqueal , Thermoplasma/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , ADN de Archaea , Endopeptidasas/metabolismo , Metabolismo Energético , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Recombinación Genética , Sulfolobus/genética , Thermoplasma/metabolismo , Ubiquitinas/metabolismoRESUMEN
Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding.
Asunto(s)
Archaea/química , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiología , Secuencia de Aminoácidos , Western Blotting , Dicroismo Circular , Proteínas HSP70 de Choque Térmico/química , Methanobacterium/química , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins.
Asunto(s)
Archaea/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pliegue de Proteína , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Codón , Estabilidad de Enzimas , Hidrólisis , Cinética , Datos de Secuencia Molecular , Penicilinasa/química , Penicilinasa/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteína que Contiene ValosinaRESUMEN
We applied a succession of sequence search and structure prediction methods to the targets in the fold recognition part of the CASP3 experiment. For each target, we expanded an initial sequence space, obtained through PSI-BLAST, by searching for statistically significant relationships to low-scoring sequences and then by searching for conserved sequence patterns. We then divided the proteins in the sequence space into families and built an alignment hierarchically, using the multiple alignment program MACAW. If no significant similarity to a protein of known structure was apparent at this point, we submitted the alignment to the Jpred server for consensus secondary structure prediction and searched the structure space using the secondary structure mapping program MAP. Failing this, we compared the structural properties that we believed we recognized in the aligned proteins to the folds in the SCOP database, using visual inspection. If all these methods failed to uncover a plausible match, we predicted that the target would adopt a novel fold. This procedure yielded correct answers for seven of twenty-one targets and a partly correct answer for one. A retrospective analysis shows that automating the sequence search procedures would have represented a significant improvement, with at least three additional correct predictions.
Asunto(s)
Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas/química , Algoritmos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
A general paradigm for energy-dependent proteases is emerging: ATP may be used to unfold the substrate and translocate it through a narrow channel within the enzyme into a central proteolytic chamber. Different members of the family present intriguing elaborations on this model.
Asunto(s)
Adenosina Trifosfato , Serina Endopeptidasas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Serina Endopeptidasas/química , Relación Estructura-ActividadAsunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas de Unión al GTP/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al GTP/fisiología , Datos de Secuencia Molecular , Relación Estructura-Actividad , Quinasas de Receptores Adrenérgicos betaRESUMEN
We used a computer-based prediction algorithm to identify probable coiled-coil segments at the N-termini of G protein alpha, beta and gamma subunits. This result indicates that G protein trimers may form via a three-stranded coiled coil. Previous biochemical results had shown that the N-termini of alpha and beta are involved in subunit interactions. Here we present a structural model for the N-terminal domain of beta gamma and a hypothesis for the reversible association of alpha to beta gamma.
Asunto(s)
Algoritmos , Proteínas de Unión al GTP/química , Modelos Moleculares , Secuencia de Aminoácidos , Proteínas de Unión al GTP/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Transducina/química , Transducina/metabolismoRESUMEN
We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.