RESUMO
Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution.
Assuntos
Evolução Molecular , Mesorhizobium/metabolismo , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Antitoxinas/química , Antitoxinas/metabolismo , Bactérias/química , Bactérias/classificação , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência MolecularRESUMO
The Alphaproteobacteria uniquely integrate features of two-component signal transduction and alternative σ factor regulation to control transcription of genes that ensure growth and survival across a range of stress conditions. Research over the past decade has led to the discovery of the key molecular players of this general stress response (GSR) system, including the sigma factor σ(EcfG), its anti-σ factor NepR, and the anti-anti-σ factor PhyR. The central molecular event of GSR activation entails aspartyl phosphorylation of PhyR, which promotes its binding to NepR and thereby releases σ(EcfG) to associate with RNAP and direct transcription. Recent studies are providing a new understanding of complex, multilayered sensory networks that activate and repress this central protein partner switch. This review synthesizes our structural and functional understanding of the core GSR regulatory proteins and highlights emerging data that are defining the systems that regulate GSR transcription in a variety of species.
Assuntos
Alphaproteobacteria/fisiologia , Proteínas de Bactérias/metabolismo , Estresse Fisiológico/fisiologia , Proteínas de Bactérias/genética , Cromossomos Bacterianos , Regulação Bacteriana da Expressão Gênica , Transdução de SinaisRESUMO
Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Myxococcus xanthus/fisiologia , Proteínas de Bactérias/genética , Polaridade Celular/fisiologia , Myxococcus xanthus/citologia , Myxococcus xanthus/genética , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do GenomaRESUMO
Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.
Assuntos
Locomoção , Myxococcus xanthus/enzimologia , Proteínas ras/metabolismo , Regulação Alostérica , Sítios de Ligação , Polaridade Celular , Conformação ProteicaRESUMO
Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.
Assuntos
Quimiotaxia , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Myxococcus xanthus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Movimento Celular , Regulação Bacteriana da Expressão Gênica , Proteínas Quimiotáticas Aceptoras de Metil/genética , Myxococcus xanthus/genética , Óperon , Fenótipo , Transdução de SinaisRESUMO
Cell growth is determined by substrate availability and the cell's metabolic capacity to assimilate substrates into building blocks. Metabolic genes that determine growth rate may interact synergistically or antagonistically, and can accelerate or slow growth, depending on genetic background and environmental conditions. We evolved a diverse set of Escherichia coli single-gene deletion mutants with a spectrum of growth rates and identified mutations that generally increase growth rate. Despite the metabolic differences between parent strains, mutations that enhanced growth largely mapped to core transcription machinery, including the ß and ß' subunits of RNA polymerase (RNAP) and the transcription elongation factor, NusA. The structural segments of RNAP that determine enhanced growth have been previously implicated in antibiotic resistance and in the control of transcription elongation and pausing. We further developed a computational framework to characterize how the transcriptional changes that occur upon acquisition of these mutations affect growth rate across strains. Our experimental and computational results provide evidence for cases in which RNAP mutations shift the competitive balance between active transcription and gene silencing. This study demonstrates that mutations in specific regions of RNAP are a convergent adaptive solution that can enhance the growth rate of cells from distinct metabolic states.
Assuntos
Adaptação Fisiológica/genética , Evolução Biológica , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Mutação , Meios de Cultura , Genes Bacterianos , TranscriptomaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1007284.].
RESUMO
Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.
Assuntos
Brucella abortus/crescimento & desenvolvimento , Brucella abortus/patogenicidade , Ciclo Celular , Parede Celular/metabolismo , Antígenos O/metabolismo , Proteínas Periplásmicas/metabolismo , Fatores de Virulência/metabolismo , Animais , Brucella abortus/enzimologia , Brucella abortus/genética , Brucella ovis/genética , Brucella ovis/crescimento & desenvolvimento , Brucelose/microbiologia , Brucelose/patologia , Modelos Animais de Doenças , Deleção de Genes , Técnicas de Silenciamento de Genes , Genes Bacterianos , Genes Essenciais , Histocitoquímica , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Conformação Proteica , Dobramento de Proteína , Baço/patologia , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
The Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space of Brucella species, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded ß-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of Brucella abortus infection. Transposon disruption of ttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal with eipB deletion. ttpA is a reported virulence determinant in Brucella, and our studies of ttpA deletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.IMPORTANCEBrucella species cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel ß protein that is a determinant of cell envelope integrity in vitro and virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.
Assuntos
Membrana Externa Bacteriana/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Periplasma/química , Animais , Brucella abortus/química , Brucelose/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Virulência , Fatores de Virulência/genéticaRESUMO
The general stress response sigma factor σE1 directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection in the zoonotic pathogen Brucella abortus Characterizing the functions of σE1-regulated genes therefore would contribute to our understanding of B. abortus physiology and infection biology. σE1 indirectly activates transcription of the IclR family regulator Bab2_0215, but the function of this regulator remains undefined. Here, we present a structural and functional characterization of Bab2_0215, which we have named B rucella adipic acid-activated regulator (BaaR). We found that BaaR adopts a classic IclR-family fold and directly represses the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and it represses transcription of both genes. We identified three carboxylic acids (adipic acid, tetradecanedioic acid, and ϵ-aminocaproic acid) and a lactone (ϵ-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhanced transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid required the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a suite of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program in B. abortus that is downstream of σE1.
Assuntos
Proteínas de Bactérias/fisiologia , Brucella abortus/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Proteínas Repressoras/fisiologia , Transcrição Gênica/genética , Adipatos/farmacologia , Ácido Aminocaproico/farmacologia , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Brucella abortus/genética , Brucella abortus/crescimento & desenvolvimento , Caproatos/farmacologia , Cromossomos Bacterianos , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Peróxido de Hidrogênio/metabolismo , Lactonas/farmacologia , Ácido Mirístico/farmacologia , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Dobramento de Proteína , Fator sigma/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.
Assuntos
Brucella abortus/fisiologia , Divisão Celular , Transdução de Sinais , Virulência , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Brucella abortus/citologia , Brucella abortus/patogenicidade , Humanos , Macrófagos/microbiologia , FosforilaçãoRESUMO
Brucella abortus σE1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the σE1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σS in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σE1-null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li+ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers.IMPORTANCEBrucella abortus σE1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σE1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σE1 Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Betaína , Transporte Biológico , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Óperon/fisiologia , FilogeniaRESUMO
Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.
Assuntos
Proteínas de Bactérias/metabolismo , Droseraceae/metabolismo , Periplasma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Bordetella pertussis/metabolismo , Cristalografia por Raios X , Droseraceae/química , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Transdução de Sinais/fisiologia , Fatores de Transcrição/química , VirulênciaRESUMO
In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA) that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ). Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.
Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/crescimento & desenvolvimento , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Nutrigenômica/métodos , Regiões Promotoras Genéticas , Transdução de Sinais/genéticaRESUMO
UNLABELLED: The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. IMPORTANCE: Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Bactérias/genética , Brucella abortus/genética , Linhagem Celular , Feminino , Humanos , Macrófagos/microbiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Gravidez , Conformação ProteicaRESUMO
Enterotoxigenic Bacteroides fragilis produces a secreted metalloprotease known as B. fragilis toxin (BFT), which contributes to anaerobic sepsis, colitis, and colonic malignancy in mouse models of disease. A C11 family cysteine protease, fragipain (Fpn), directly activates BFT in the B. fragilis cell by removing the BFT prodomain. Fpn is itself a proenzyme and is autoactivated upon cleavage at an arginine residue in its activation loop. We have defined the proteolytic active site of Fpn, demonstrated that Fpn autoactivation can occur by an in trans loop cleavage mechanism, and characterized structural features of the Fpn activation loop that control peptidase activity against several substrates, including BFT. An arginine residue at the autocleavage site determines the fast activation kinetics of Fpn relative to the homologous C11 protease, PmC11, which is cleaved at lysine. Arginine to alanine substitution at the cleavage site ablated peptidase activity, as did partial truncation of the Fpn activation loop. However, complete truncation of the activation loop yielded an uncleaved, pro form of Fpn that was active as a peptidase against both Fpn and BFT substrates. Thus, Fpn can be transformed into an active peptidase in the absence of activation loop cleavage. This study provides insight into the mechanism of fragipain activation and, more generally, defines the role of the C11 activation loop in the control of peptidase activity and substrate specificity.
Assuntos
Toxinas Bacterianas/metabolismo , Bacteroides fragilis/enzimologia , Cisteína Proteases/metabolismo , Metaloendopeptidases/metabolismo , Substituição de Aminoácidos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Bacteroides fragilis/genética , Bacteroides fragilis/patogenicidade , Domínio Catalítico , Cisteína Proteases/química , Cisteína Proteases/genética , Ativação Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Cinética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
α-Proteobacteria uniquely integrate features of two-component signal transduction (TCS) and alternative sigma factor (σ) regulation to control transcription in response to general stress. The core of this regulatory system is the PhyR protein, which contains a σ-like (SL) domain and a TCS receiver domain. Aspartyl phosphorylation of the PhyR receiver in response to stress signals promotes binding of the anti-σ factor, NepR, to PhyR-SL. This mechanism, whereby NepR switches binding between its cognate σ factor and phospho-PhyR (PhyRâ¼P), controls transcription of the general stress regulon. We have defined the structural basis of the PhyRâ¼P/NepR interaction in Caulobacter crescentus and characterized the effect of aspartyl phosphorylation on PhyR structure by molecular dynamics simulations. Our data support a model in which phosphorylation of the PhyR receiver domain promotes its dissociation from the PhyR-SL domain, which exposes the NepR binding site. A highly dynamic loop-helix region (α3-α4) of the PhyR-SL domain plays an important role in PhyRâ¼P binding to NepR in vitro, and in stress-dependent activation of transcription in vivo. This study provides a foundation for understanding the protein-protein interactions and protein structural dynamics that underpin general stress adaptation in a large and metabolically diverse clade of the bacterial kingdom.
Assuntos
Alphaproteobacteria/genética , Caulobacter crescentus/genética , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico/genética , Alphaproteobacteria/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Cristalografia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/fisiologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator sigma/antagonistas & inibidores , Fator sigma/química , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Transcrição Gênica/fisiologiaRESUMO
As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7â nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Pareamento de Bases , Cristalografia por Raios X , Modelos MolecularesRESUMO
Myxococcus relies on motility to efficiently invade and predate a prey colony. Upon contact with prey, Myxococcus temporarily halts its motility and initiates prey cell lysis, which involves two contact-dependent predatory machineries, the Kil system and the needleless T3SS*. Predatory cells grow as they invade and feed on prey cells. When dividing, Myxococcus cells systematically pause their movements before division. This highlights a high level of co-ordination between motility and contact-dependent killing but also with cell division. In this review, we give an overview of the different nanomachines used by Myxococcus to move on surfaces, kill by contact, and divide, and we discuss the potential regulatory mechanisms at play during these different processes.
Assuntos
Myxococcus , Myxococcus/genética , Myxococcus/fisiologia , Divisão Celular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
The periplasmic binding protein (PBP) IbpA mediates the uptake of myo-inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo-inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-ß lobes that surround a central hinge. A pocket positioned between the lobes contains the myo-inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 µM). IbpA is homologous to ribose-binding proteins and binds D-ribose with low affinity (50.8 ± 3.4 µM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo-inositol and D-ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for D-ribose by 10-fold while completely abolishing binding to myo-inositol. Replacement of ibpA with these mutant alleles unable to bind myo-inositol abolishes C. crescentus growth in medium containing myo-inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on D-ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo-inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands.