RESUMO
The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determined six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme and phosphodiesterase separated by nearly 180 Å. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels.
RESUMO
FixL is a prototype for heme-based sensors, multidomain proteins that typically couple a histidine protein kinase activity to a heme-binding domain for sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. Despite the relatively well-developed understanding of FixL, the importance of some of its domains has been unclear. To explore the impact of domain-domain interactions on oxygen sensing and signal transduction, we characterized and investigated Rhizobium etli hybrid sensor ReFixL. In ReFixL, the core heme-containing PAS domain and kinase region is preceded by an N-terminal PAS domain of unknown function and followed by a C-terminal receiver domain. The latter resembles a target substrate domain that usually occurs independently of the kinase and contains a phosphorylatable aspartate residue. We isolated the full-length ReFixL as a soluble holoprotein with a single heme b cofactor. Despite a low affinity for oxygen (K(d) for O2 of 738 µM), the kinase activity was completely switched off by O2 at concentrations well below the K(d). A deletion of the first PAS domain strongly increased the oxygen affinity but essentially prohibited autophosphorylation, although the truncated protein was competent to accept phosphoryl groups in trans. These studies provide new insights into histidyl-aspartyl phosphoryl transfers in two-component systems and suggest that the control of ligand affinity and signal transduction by PAS domains can be direct or indirect.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Bactérias/metabolismo , Hemeproteínas/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Heme/metabolismo , Hemeproteínas/química , Hemeproteínas/genética , Histidina/metabolismo , Histidina Quinase , Cinética , Ligantes , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rhizobium/enzimologia , Rhizobium/metabolismoRESUMO
Since their initial discovery some 30 years ago, heme-based O2 sensors have been extensively studied. Among many other lessons, we have learned that they have adapted a wide variety of folds to bind heme for O2 sensing, and they can couple those sensory domains to transducer domains with many different activities. There is no question that we have learned a great deal about those systems by solving X-ray structures of the truncated pieces of larger multi-domain proteins. All of the studies have, for example, hinted at the importance of protein residues, which were further investigated, usually by site-directed mutagenesis of the full-length proteins together with physico-chemical measurements and enzymatic studies. The biochemistry has suggested that the sensing functions of heme-based O2 sensors involve not only the entire proteins but also, and quite often, their associated regulatory partners and targets. Here we critically examine the state of knowledge for some well-studied sensors and discuss outstanding questions regarding their structures. For the near future, we may foresee many large complexes with sensor proteins being solved by cryo-EM, to enhance our understanding of their mechanisms.
Assuntos
Heme , Hemeproteínas , Heme/química , Oxigênio/química , Hemeproteínas/química , Proteínas de Bactérias/químicaRESUMO
The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.
Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Fotossíntese/fisiologia , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dados de Sequência Molecular , Fotossíntese/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Rhodobacter sphaeroides/genética , Homologia de Sequência de AminoácidosRESUMO
A commonly observed coupling of sensory domains to GGDEF-class diguanylate cyclases and EAL-class phosphodiesterases has long suggested that c-di-GMP synthesizing and degrading enzymes sense environmental signals. Nevertheless, relatively few signal ligands have been identified for these sensors, and even fewer instances of in vitro switching by ligand have been demonstrated. Here we describe an Escherichia coli two-gene operon, dosCP, for control of c-di-GMP by oxygen. In this operon, the gene encoding the oxygen-sensing c-di-GMP phosphodiesterase Ec Dos (here renamed Ec DosP) follows and is translationally coupled to a gene encoding a diguanylate cyclase, here designated DosC. We present the first characterizations of DosC and a detailed study of the ligand-dose response of DosP. Our results show that DosC is a globin-coupled sensor with an apolar but accessible heme pocket that binds oxygen with a K(d) of 20 microM. The response of DosP activation to increasing oxygen concentration is a complex function of its ligand saturation such that over 80% of the activation occurs in solutions that exceed 30% of air saturation (oxygen >75 microM). Finally, we find that DosP and DosC associate into a functional complex. We conclude that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.
Assuntos
GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/fisiopatologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bordetella pertussis/enzimologia , Bordetella pertussis/metabolismo , GMP Cíclico/química , GMP Cíclico/isolamento & purificação , GMP Cíclico/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Óperon/genética , Oxigênio/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/isolamento & purificação , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/isolamento & purificação , Ligação ProteicaRESUMO
The Escherichia coli operon dosCP, also called yddV-yddU, co-expresses two heme proteins, DosC and DosP, both of which are direct oxygen sensors but paradoxically have opposite effects on the levels of the second messenger c-di-GMP. DosC is a diguanylate cyclase that synthesizes c-di-GMP from GTP, whereas DosP is a phosphodiesterase that linearizes c-di-GMP to pGpG. Both proteins are associated with the large degradosome enzyme complex that regulates many bacterial genes post-transcriptionally by processing or degrading the corresponding RNAs. Moreover, the c-di-GMP directly binds to PNPase, a key degradosome enzyme, and enhances its activity. This review combines biochemical, biophysical, and genetic findings on DosC and DosP, a task that has not been undertaken until now, partly because of the varied nomenclature. The DosC and DosP system is examined in the context of the current knowledge of degradosomes and considered as a possible prototype for the compartmentalization of sensing by E. coli.
Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nucleotidiltransferases/metabolismo , Diester Fosfórico Hidrolases/metabolismo , GMP Cíclico/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/genética , Diester Fosfórico Hidrolases/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Sistemas do Segundo MensageiroRESUMO
A major challenge to the control and eventual eradication of Mycobacterium tuberculosis infection is this pathogen's prolonged dormancy. The heme-based oxygen sensor protein DevS (DosS) plays a key role in this phenomenon, because it is a major activator of the transcription factor DevR. When DevS is active, its histidine protein kinase region is ON and it phosphorylates and activates DevR, which can induce the transcription of the dormancy regulon genes. Here, we have investigated the mechanism by which the ligation of molecular oxygen to a heme-binding domain in DevS switches OFF its histidine protein kinase region. To shed light on the oligomerization states of this protein and possible protein-surfaces of interaction, we used analytical gel filtration, together with dynamic light scattering, fluorescence spectroscopy and chemical crosslinking. We found that DevS exists as three major species: an octamer, a tetramer and a dimer. These three states were observed for the concentration range between 0.5 and 20 µm DevS, but not below 0.1 µm. Levels of DevS in M. tuberculosis are expected to range from 5 to 26 µm. When this histidine protein kinase was OFF, the DevS was mainly tetrameric and dimeric; by contrast, when the kinase was ON, the protein was predominantly octameric. The changes in quaternary structure were rapid upon binding to the physiological signal. This finding represents a novel strategy for switching the activity of a two-component heme-based sensor. An enhanced understanding of this process might potentially lead to the design of novel regulatory agents that target the multimer interfaces for treatment of latent tuberculosis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Heme/química , Mycobacterium tuberculosis/efeitos dos fármacos , Oxigênio/farmacologia , Protamina Quinase/química , Proteínas Quinases/genética , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Clonagem Molecular , Proteínas de Ligação a DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulon , Transdução de Sinais , Espectrometria de Fluorescência , Transcrição Gênica/efeitos dos fármacosRESUMO
Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the K(d) values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (k(cat)) of the protein substrate by sensors of known heme status.
Assuntos
Oxigênio/análise , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Hemeproteínas/análise , Hemeproteínas/fisiologia , Histidina Quinase , Ligantes , Oxigênio/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/isolamento & purificação , Controle de Qualidade , Titulometria/métodosRESUMO
Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain. For full-length DosT and DevS, we determined the ligand-binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO-bound, and NO-bound forms were active, but the O(2)-bound form was inactive. Oxygen-bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O(2) to DosT (K(d) [NO] approximately 5 nM versus K(d) [O(2)] = 26 microM). These results demonstrate direct and specific O(2) sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O(2) binding and yielding an active kinase.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/enzimologia , Oxigênio/metabolismo , Protamina Quinase/química , Proteínas Quinases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Heme/química , Heme/metabolismo , Cinética , Ligantes , Modelos Biológicos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Protamina Quinase/isolamento & purificação , Protamina Quinase/metabolismo , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Estrutura Terciária de ProteínaRESUMO
In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd approximately 350 microM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the Fe(III) form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.
Assuntos
Arginina/química , Proteínas de Bactérias/química , Bradyrhizobium/metabolismo , Hemeproteínas/química , Oxigênio/metabolismo , Sequência de Aminoácidos , Escherichia coli/metabolismo , Histidina/química , Histidina Quinase , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Haem-based sensors have emerged during the last 15 years as being a large family of proteins that occur in all kingdoms of life. These sensors are responsible mainly for detecting binding of O2, CO and NO and reporting the ligation status to an output domain with an enzymatic or macromolecule-binding property. A myriad of biological functions have been associated with these sensors, which are involved in vasodilation, bacterial symbiosis, chemotaxis and biofilm formation, among others. Here, we critically review several bacterial systems for O2 sensing that are extensively studied in many respects, focusing on the lessons that are important to advance the field.
Assuntos
Heme/fisiologia , Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Heme/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , Rhizobium/metabolismoRESUMO
Mycobacterium tuberculosis strongly relies on a latency, or nonreplicating persistence, to escape a human host's immune system. The DevR (DosR), DevS (DosS), and DosT proteins are key components of this process. Like the rhizobial FixL oxygen sensor, DevS and DosT are histidine protein kinases with a heme-binding domain. Like the FixJ partner and substrate of FixL, DevR is a classical response regulator of the two-component class. When activated by DevS or DosT during hypoxia in vivo, DevR induces a dormancy regulon of more than 40 genes. To investigate the contributions of DevS, DosT, and target DNA to the phosphorylation of DevR, we developed an in vitro assay in which the full-length, sensing, DevS and DosT proteins were used to phosphorylate DevR with ATP, in the presence of target DNAs that were introduced as oligonucleotides linked to magnetic nanoparticles. We found that the DevR phosphorylations proceeded only for the deoxy states of the sensors. The reaction was strongly inhibited by O2 , but not CO or NO. The production of phospho-DevR was enhanced sixfold by target consensus DNA or acr-DNA. The phospho-DevR bound tightly to that DNA (Kd ~ 0.8 nm toward acr-DNA), and it was only slightly displaced by a 200-fold excess of unphosphorylated DevR or of a truncated DevR with only a DNA-binding domain. To our knowledge, this represents the first in vitro study of the ligand regulation of DevR phosphorylation by full-length DevS and DosT, and demonstration of a positive effect of DNA on this reaction.
Assuntos
Proteínas de Bactérias/metabolismo , DNA/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxigênio/metabolismo , Protamina Quinase/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Bactérias/química , DNA/química , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica , Humanos , Nanopartículas de Magnetita/química , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fosforilação , Protamina Quinase/química , Proteínas Quinases/química , RegulonRESUMO
Heme-based sensors have emerged during the last 20years as being a large family of proteins that occur in all kingdoms of life. A myriad of biological adaptations are associated with these sensors, which include vasodilation, bacterial virulence, dormancy, chemotaxis, biofilm formation, among others. Due to the key activities regulated by these proteins along with many other systems that use similar output domains, there is a growing interest in developing small molecules as their regulators. Here, we review the development of potential activators and inhibitors for many of these systems, including human soluble guanylate cyclase, c-di-GMP-related enzymes, Mycobacterium tuberculosis DevR/DevS/DosT (differentially expressed in virulent strain response regulator/sensor/dormancysurvival sensorT), the Rev-erb-α and ß nuclear receptor, among others. The possible roles of these molecules as biochemical tools, therapeutic agents, and novel antibiotics are critically examined.
Assuntos
Antibacterianos , Técnicas Biossensoriais/métodos , Descoberta de Drogas/métodos , Mycobacterium tuberculosis , Guanilil Ciclase Solúvel/química , Animais , Antibacterianos/análise , Antibacterianos/síntese química , Antibacterianos/química , Heme , HumanosRESUMO
FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(FeIII) and inactive cyanomet-FixL (FeIII-CN-) form was monitored by UV-visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV-visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔGH2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8kJmol-1). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2kJmol-1). For the three-state mechanism exhibited by fluorescence, the ΔGH2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.
Assuntos
Proteínas de Bactérias/metabolismo , Hemeproteínas/metabolismo , Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Fluorescência , Histidina Quinase , Oxigênio/química , Desnaturação Proteica , Dobramento de Proteína , Análise Espectral , Ureia/químicaRESUMO
In a great variety of organisms throughout all kingdoms of life, the heme-based-sensor proteins are the key regulators of adaptive responses to fluctuating oxygen, carbon monoxide, and nitric oxide levels. These signal transducers achieve their responses by coupling a regulatory heme-binding domain to a neighboring transmitter. The past decade has witnessed an explosion in the numbers of these modular sensory proteins known, from just two recognized members, FixL and soluble guanylyl cyclase (sGC), to four broad families comprising more than 50 sensors. Heme-based sensors so far feature four different types of heme-binding modules: the heme-binding PAS domain, globin-coupled sensor (GCS), CooA, and heme-NO-binding (HNOB). The transmitters for coupling to such heme-binding domains include histidine protein kinases, cyclic nucleotide phosphodiesterases, chemotaxis methyl-carrier protein receptors, and transcription factors of the basic helix-loop-helix and helix-turn-helix classes. Some well-studied sensors are the FixL, EcDos, AxPDEA1, NPAS2, HemAT-Bs, HemAT-Hs, CooA, and sGC proteins. This review elaborates the defining characteristics of heme-based sensors, examines recent developments on those proteins, and discusses the regulatory hypotheses proposed for those sensors. A general, "helix-swap", model is also proposed here for signal transduction by PAS domains.
Assuntos
Hemeproteínas/química , Hemeproteínas/metabolismo , Óxido Nítrico/química , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Técnicas Biossensoriais , Monóxido de Carbono/química , Hemeproteínas/classificação , Hemeproteínas/genética , Modelos Moleculares , Oxirredução , Oxigênio/química , Filogenia , Ligação Proteica , Conformação ProteicaRESUMO
The most common physiological strategy for detecting the gases oxygen, carbon monoxide, and nitric oxide is signal transduction by heme-based sensors, a broad class of modular proteins in which a heme-binding domain governs the activity of a neighboring transmitter domain. Different structures are possible for the heme-binding domains in these sensors, but, so far, the Per-ARNT-Sim motif, or PAS domain, is the one most commonly encountered. Heme-binding PAS (heme-PAS) domains can accomplish ligand-dependent switching of a variety of partner domains, including histidine kinase, phosphodiesterase, and basic helix-loop-helix (bHLH) DNA-binding modules. Proteins with heme-PAS domains occur in all kingdoms of life and are quite diverse in their physiological roles. Examples include the neuronal bHLH-PAS carbon monoxide sensor NPAS2 that is implicated in the mammalian circadian clock, the acetobacterial oxygen sensor AxPDEA1 that directs cellulose production, and the rhizobial oxygen sensor FixL, which governs nitrogen fixation. What factors determine the range of detection of these sensors? How do they transduce their signal? This review examines the recent advances in answering these questions.
Assuntos
Proteínas de Bactérias/fisiologia , Células Quimiorreceptoras/fisiologia , Hemeproteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas de Bactérias/química , Células Quimiorreceptoras/química , Hemeproteínas/química , Estrutura Terciária de Proteína , Receptores de Superfície CelularRESUMO
Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O(2)] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O(2) with moderate affinity, 135 and 222 µM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O(2)-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Oxigênio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Chlamydomonas reinhardtii/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Hemeproteínas/química , Hemeproteínas/genética , Histidina/química , Histidina Quinase , Dados de Sequência Molecular , Mutação , Fosforilação , EspectrofotometriaRESUMO
The second messenger cyclic diguanylic acid (c-di-GMP) is implicated in key lifestyle decisions of bacteria, including biofilm formation and changes in motility and virulence. Some challenges in deciphering the physiological roles of c-di-GMP are the limited knowledge about the cellular targets of c-di-GMP, the signals that control its levels, and the proportion of free cellular c-di-GMP, if any. Here, we identify the target and the regulatory signal for a c-di-GMP-responsive Escherichia coli ribonucleoprotein complex. We show that a direct c-di-GMP target in E. coli is polynucleotide phosphorylase (PNPase), an important enzyme in RNA metabolism that serves as a 3' polyribonucleotide polymerase or a 3'-to-5' exoribonuclease. We further show that a complex of polynucleotide phosphorylase with the direct oxygen sensors DosC and DosP can perform oxygen-dependent RNA processing. We conclude that c-di-GMP can mediate signal-dependent RNA processing and that macromolecular complexes can compartmentalize c-di-GMP signaling.
Assuntos
GMP Cíclico/análogos & derivados , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , GMP Cíclico/metabolismo , Endorribonucleases/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Substâncias Macromoleculares , Oxigênio/metabolismo , Fosfopiruvato Hidratase/metabolismo , RNA/genéticaRESUMO
Hell's Gate globin I (HGbI), a heme-containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O(2) and shows barely detectable autoxidation in the pH range of 5.2-8.6 and temperature range of 25-50°C. Examination of the heme pocket by X-ray crystallography and molecular dynamics showed that conformational movements of Tyr29(B10) and Gln50(E7), as well as structural flexibility of the GH loop and H-helix, may play a role in modulating its ligand binding behavior. Bacterial HGbI's unique resistance to the sort of extreme acidity that would extract heme from any other hemoglobin makes it an ideal candidate for comparative structure-function studies of the expanding globin superfamily.
Assuntos
Proteínas de Bactérias/química , Bactérias Gram-Negativas/química , Hemoglobinas/química , Cristalografia por Raios X , Globinas/química , Humanos , Concentração de Íons de Hidrogênio , Proteínas do Tecido Nervoso/química , Neuroglobina , Oxigênio/química , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
Globin-coupled sensors are heme-binding signal transducers in Bacteria and Archaea in which an N-terminal globin controls the activity of a variable C-terminal domain. Here, we report that BpeGReg, a globin-coupled diguanylate cyclase from the whooping cough pathogen Bordetella pertussis, synthesizes the second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) upon oxygen binding. Expression of BpeGReg in Salmonella typhimurium enhances biofilm formation, while knockout of the BpeGReg gene of B. pertussis results in decreased biofilm formation. These results represent the first identification a signal ligand for any diguanylate cyclase and provide definitive experimental evidence that a globin-coupled sensor regulates c-di-GMP synthesis and biofilm formation. We propose that the synthesis of c-di-GMP by globin sensors is a widespread phenomenon in bacteria.