RESUMO
La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].
Assuntos
Poli A , Ligação Proteica , Ribonucleoproteínas , Antígeno SS-B , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Poli A/metabolismo , Poli A/química , Humanos , Modelos Moleculares , Sítios de Ligação , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Cristalografia por Raios X , Domínios Proteicos , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , RNA Mensageiro/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O2-dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O2 binding modulates activity of diguanylate cyclase-containing GCS proteins.
Assuntos
Proteínas de Bactérias , Heme , Pectobacterium carotovorum , Fósforo-Oxigênio Liases , Fósforo-Oxigênio Liases/metabolismo , Fósforo-Oxigênio Liases/química , Heme/química , Heme/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pectobacterium carotovorum/enzimologia , Conformação Proteica , Oxigênio/química , Oxigênio/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Proteínas de Escherichia coliRESUMO
Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8â³ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.
Assuntos
GMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Sítios de Ligação , Domínio Catalítico , GMP Cíclico/químicaRESUMO
Protein kinase G (PKG) is an essential regulator of eukaryotic cyclic guanosine monophosphate (cGMP)-dependent intracellular signaling, controlling pathways that are often distinct from those regulated by cyclic adenosine monophosphate (cAMP). Specifically, the C-terminal cyclic-nucleotide-binding domain (CNB-B) of PKG has emerged as a critical module to control allostery and cGMP-selectivity in PKG. While key contributions to the cGMP-versus-cAMP selectivity of CNB-B were previously assessed, only limited knowledge is currently available on how cyclic nucleotide binding rewires the network of hydrogen bonds in CNB-B, and how such rewiring contributes to allostery and cGMP selectivity. To address this gap, we extend the comparative analysis of apo, cAMP- and cGMP-bound CNB-B to H/D fractionation factors (FFs), which are well-suited for assessing backbone hydrogen-bond strengths within proteins. Apo-vs-bound comparisons inform of perturbations arising from both binding and allostery, while cGMP-bound vs cAMP-bound comparisons inform of perturbations that are purely allosteric. The comparative FF analyses of the bound states revealed mixed patterns of hydrogen-bond strengthening and weakening, pointing to inherent frustration, whereby not all hydrogen bonds can be simultaneously stabilized. Interestingly, contrary to expectations, these patterns include a weakening of hydrogen bonds not only within critical recognition and allosteric elements of CNB-B, but also within elements known to undergo rigid-body movement upon cyclic nucleotide binding. These results suggest that frustration may contribute to the reversibility of allosteric conformational shifts by avoiding over-rigidification that may otherwise trap CNB-B in its active state. Considering that PKG CNB-B serves as a prototype for allosteric conformational switches, similar concepts may be applicable to allosteric domains in general.
Assuntos
AMP Cíclico , Nucleotídeos Cíclicos , Nucleotídeos Cíclicos/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Ligação Proteica , HidrogênioRESUMO
BACKGROUND: Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na+) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na+ transport similar to ouabain-a cardiotonic steroid. METHODS: Urine Na+ excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 µg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA. RESULTS: Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated SrcTyr416 and Erk 1/2 (extracellular signal-regulated protein kinase 1/2)Thr202/Tyr204; these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 µM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 µM)+ouabain (10 µM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N3-PET-8-biotin-11-cGMP (2 µM); 8-N3-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N3-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N3-6-biotin-10-cAMP RPTs. Ouabain (10 µM) reduced NKA in cross-linked 4-N3-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N3-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. CONCLUSIONS: cGMP can bind to NKA and thereby mediate natriuresis.
Assuntos
GMP Cíclico , Natriurese , ATPase Trocadora de Sódio-Potássio , Animais , Feminino , Ratos , Adenosina Trifosfatases/metabolismo , Biotina/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Natriurese/fisiologia , Ouabaína/farmacologia , Potássio/metabolismo , Ratos Sprague-Dawley , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Cyclic Nucleotides are important in regulating platelet function. Increases in 3'5'-cyclic adenosine monophosphate (cAMP) and 3'5'-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.
Assuntos
AMP Cíclico , GMP Cíclico , Cromatografia Líquida/métodos , GMP Cíclico/análise , GMP Cíclico/química , AMP Cíclico/análise , Espectrometria de Massas em Tandem/métodos , Agregação Plaquetária , Plaquetas/químicaRESUMO
Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.
Assuntos
Cianobactérias , Guanosina Monofosfato , Proteínas de Bactérias/genética , Cromatografia Líquida/métodos , Cianobactérias/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/análise , GMP Cíclico/química , GMP Cíclico/metabolismo , Guanosina Monofosfato/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem/métodosRESUMO
The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.
Assuntos
Proteínas de Membrana/agonistas , Animais , Cristalografia por Raios X , AMP Cíclico/química , AMP Cíclico/farmacologia , GMP Cíclico/química , GMP Cíclico/farmacologia , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Imunoterapia/métodos , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Neoplasias/imunologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in ß-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.
Assuntos
Bactérias/metabolismo , Imunidade Inata , Proteínas de Membrana/química , Cristalografia por Raios X , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Fosfatos de Dinucleosídeos , Humanos , Interferons , Ligantes , Proteínas de Membrana/genética , Nucleotidiltransferases/metabolismo , PrevotellaRESUMO
Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.
Assuntos
Proteínas de Bactérias/química , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/química , Heme/química , Hemeproteínas/química , Paenibacillus/química , Diester Fosfórico Hidrolases/química , Fósforo-Oxigênio Liases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , GMP Cíclico/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Heme/metabolismo , Hemeproteínas/genética , Hemeproteínas/metabolismo , Cinética , Modelos Moleculares , Oxigênio/química , Oxigênio/metabolismo , Paenibacillus/enzimologia , Paenibacillus/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Recent improvements in mRNA display have enabled the selection of peptides that incorporate non-natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non-natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino-acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non-natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25-letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING-binding peptides include a 9-mer macrocycle with a dissociation constant (KD ) of 3.4â nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino-acid alphabet in a library beyond 25 building blocks.
Assuntos
Proteínas de Membrana/metabolismo , Peptídeos Cíclicos/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Códon , AMP Cíclico/química , AMP Cíclico/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Dimerização , Engenharia Genética , Humanos , Cinética , Proteínas de Membrana/química , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , RNA Mensageiro/genéticaRESUMO
Biofilms, the predominant growth mode of microorganisms, pose a significant risk to human health. The protective biofilm matrix, typically composed of exopolysaccharides, proteins, nucleic acids, and lipids, combined with biofilm-grown bacteria's heterogenous physiology, leads to enhanced fitness and tolerance to traditional methods for treatment. There is a need to identify biofilm inhibitors using diverse approaches and targeting different stages of biofilm formation. This review discusses discovery strategies that successfully identified a wide range of inhibitors and the processes used to characterize their inhibition mechanism and further improvement. Additionally, we examine the structure-activity relationship (SAR) for some of these inhibitors to optimize inhibitor activity.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Antibacterianos/biossíntese , Antibacterianos/síntese química , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/química , GMP Cíclico/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Farmacorresistência Bacteriana/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/patogenicidade , Lipídeos/antagonistas & inibidores , Lipídeos/química , Testes de Sensibilidade Microbiana , Ácidos Nucleicos/antagonistas & inibidores , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Polissacarídeos Bacterianos/antagonistas & inibidores , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Protein kinase G (PKG) is a major receptor of cGMP, and controls signaling pathways distinct from those regulated by cAMP. However, the contributions of the two substituents that differentiate cGMP from cAMP (i.e. 6-oxo and 2-NH2) to the cGMP-versus-cAMP selectivity of PKG remain unclear. Here, using NMR to map how binding affinity and dynamics of the protein and ligand vary along a ligand double-substitution cycle, we show that the contributions of the two substituents to binding affinity are surprisingly non-additive. Such non-additivity stems primarily from mutual protein-ligand conformational selection, whereby not only does the ligand select for a preferred protein conformation upon binding, but also, the protein selects for a preferred ligand conformation. The 6-oxo substituent mainly controls the conformational equilibrium of the bound protein, while the 2-NH2 substituent primarily controls the conformational equilibrium of the unbound ligand (i.e. syn versus anti). Therefore, understanding the conformational dynamics of both the protein and ligand is essential to explain the cGMP-versus-cAMP selectivity of PKG.
Assuntos
AMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/química , GMP Cíclico/química , Sítios de Ligação , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Expressão Gênica , Humanos , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.
Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , GMP Cíclico/análogos & derivados , Cristalização , GMP Cíclico/química , Modelos Moleculares , Domínios ProteicosRESUMO
Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.
Assuntos
Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Vibrio parahaemolyticus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Sistemas do Segundo Mensageiro , Vibrio parahaemolyticus/genéticaRESUMO
Monophosphate, an essential component of nucleic acids, as well as cell membranes and signaling molecules, is often bound to metal cations. Despite the biological importance of monophosphate-containing cell-signaling or lipid molecules, their propensity to bind the two most abundant cellular dications, Mg2+ and Ca2+, in a particular mode (inner/outer shell, mono/bidentate) is not well understood. Whether they prefer binding to Mg2+ than to Ca2+ and if they can outcompete the carboxylates of excitatory Asp/Glu and inhibitory gamma-aminobutyric acid (GABA) neurotransmitters in binding to Mg2+/Ca2+ remain unclear. To address these questions, we modeled cyclic adenosine/guanosine monophosphate (cAMP/cGMP), nucleoside 2',3'-cyclic phosphate, phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) and determined their most stable metal-binding modes, including those of Asp/Glu and GABA, as well as their selectivity for Mg2+/Ca2+ using density functional theory combined with the polarizable continuum model. The results obtained, which are consistent with the available experimental findings, reveal that the structurally and functionally diverse monophosphate-containing ligands studied prefer monodentate coordination of Mg2+ because of the greater strain encountered upon bidentate coordination, whereas the larger Ca2+ imposes less strain upon bidentate binding and has reduced/no preference for monodentate coordination. We further show that in a low-dielectric environment, negatively charged monophosphate-containing ligands favor the better charge-accepting dication, that is, Mg2+ rather than Ca2+. By promoting Mg2+ over Ca2+ binding, signaling monophosphates (cAMP/cGMP) do not entrap cellular Ca2+ and interfere with signal transduction processes employing Ca2+ as a second messenger. In regions with high glutamate cytoplasmic concentration, glutamate may sequester Mg2+ bound to isolated five-/six-membered ring phosphates, PI, or neutral PEA, but not anionic phospholipids constituting the inner leaflet of the cell membrane.
Assuntos
Cálcio/química , AMP Cíclico/química , GMP Cíclico/química , Lipídeos/química , Magnésio/química , Modelos Moleculares , Estrutura Molecular , TermodinâmicaRESUMO
Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.
Assuntos
Alginatos/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Fímbrias/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , GMP Cíclico/química , GMP Cíclico/farmacologia , Proteínas de Fímbrias/metabolismo , Ligantes , Oxirredução , Fenótipo , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-AtividadeRESUMO
The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.
Assuntos
GMP Cíclico/química , Guanilato Ciclase/química , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Segmento Externo da Célula Bastonete/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Reagentes de Ligações Cruzadas/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Succinimidas/químicaRESUMO
New 1,3-diazaphenoxazine derivatives (nitroG-Grasp-Guanidine, NGG) have been developed to covalently capture 8-nitro-cGMP in neutral aqueous solutions, which furnish a thiol reactive group to displace the 8-nitro group and a guanidine unit for interaction with the cyclic phosphate. The thiol group was introduced to the 1,3-diazaphenoxazine skeleton through a 2-aminobenzylthiol group (NGG-H) and its 4-methyl (NGG-pMe) and 6-methyl (NGG-oMe) substituted derivatives. The covalent adducts were formed between the NGG derivatives and 8-nitro-cGMP in neutral aqueous solutions. Among the NGG derivatives, the one with the 6-methyl group (NGG-oMe) exhibited the most efficient capture reaction. Furthermore, NGG-H showed a cell permeability into HEK-293 and RAW 264.7 cells and reduced the intracellular 8-nitro-cGMP level. The NGG derivatives developed in this study would become a valuable tool to study the intracellular role of 8-nitro-cGMP.
Assuntos
GMP Cíclico/análogos & derivados , Animais , GMP Cíclico/química , GMP Cíclico/metabolismo , Células HEK293 , Humanos , Camundongos , Células RAW 264.7 , Análise Espectral/métodos , ÁguaRESUMO
Bacteria can form a biofilm composed of diverse bacterial microorganism, which work as a barrier to protect from threats, such as antibiotics and host immunity system. The formation of biofilms significantly impairs the efficacy of antibiotics against pathogenic bacteria. It is also a serious problem to be solved that the emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA) accelerated by the overuse of antibiotics. Therefore, the usage of biofilm inhibition agents has attracted immense interest as a novel strategy for treatment of diseases related to bacterial infection. From the difference of mode of action against bacterial cells, biofilm inhibition agents are expected to circumvent the emergence of multidrug-resistant bacteria. In this study, we have developed the derivatives of c-di-GMP, a kind of cyclic dinucleotide that is expected to have the effect of inhibiting bacterial biofilm formation. Some of the synthesized derivatives were found to inhibit biofilm formation of Gram-positive bacteria.