RESUMO
Protein kinase A (PKA) is a ubiquitous cAMP-dependent enzyme in mammalian tissues. The inactive PKA holoenzyme disassociates into a homodimer of regulatory (R) subunits and two active catalytic (C) subunits upon cAMP binding to two tandem domains (termed CBD-A and CBD-B) in R subunits. The release of cAMP facilitates reassociation of R and C subunits, resetting PKA to its basal state. The cAMP-mediated structural changes in the activation-termination cycle remain partially understood. The multimeric states of PKA complicate the issue and are particularly less studied. Therefore, we computationally investigated the conformational dynamics of the PKA RIα homodimer in different cAMP-bound states. The absence of cAMP in two CBDs differently affects the conformational dynamics of protomers. Moreover, such disparate responses are extended to the dimer interface constituted by the N-terminal helical sub-domains termed N3A motifs. The removal of cAMP from CBD-A induces large-scale structural changes of individual R subunits towards the holoenzyme state, consistent with previous simulations of a single R subunit. Meanwhile it keeps the structural heterogeneity of the N3A-N3A' dimer interface observed in the fully bound state. By contrast, the removal of cAMP from CBD-B does not affect individual R subunits but alters the conformational space of the N3A-N3A' dimer interface. The cAMP-coupled structural changes of each protomer and conserved conformational space of the N3A-N3A' dimer interface are essential for the transition between the fully cAMP-bound R2 homodimer and the R2C2 holoenzyme as suggested by their crystal structures. Our work provides structural insights into the regulatory mechanism of cAMP in PKA signaling.
Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , AMP Cíclico , Multimerização Proteica , AMP Cíclico/metabolismo , AMP Cíclico/química , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.
Assuntos
Adenilil Ciclases , AMP Cíclico , Deinococcus , Fotorreceptores Microbianos , Fitocromo , Proteínas Recombinantes de Fusão , Animais , Adenilil Ciclases/química , Adenilil Ciclases/genética , AMP Cíclico/química , Luz , Optogenética , Transdução de Sinais , Engenharia de Proteínas , Fitocromo/química , Fitocromo/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genéticaRESUMO
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: The clinical utility of tolvaptan in chronic kidney disease (CKD) patients with heart failure remains uncertain. The level of urine cyclic adenosine monophosphate (AMP) relative to plasma arginine vasopressin (AVP) indicates the residual function of the collecting ducts in response to AVP stimulation and might be a key to predicting response of tolvaptan. METHODS: CKD patients who were hospitalized to treat their congestive heart failure refractory to conventional loop diuretics were considered to receive tolvaptan and included in this prospective study. The impact of urine cyclic AMP/plasma AVP ratio for prediction of response to tolvaptan, which was defined as any increase in urine volume at day 7 from day 0, was investigated. RESULTS: A total of 30 patients (median 75 years old, 24 men, and median estimated glomerular filtration rate 14.4 mL/min/1.73 m2) were included. As compared to baseline, urine volume increased at day 7 in 17 responders, whereas urine volume decreased at day 7 in 13 non-responders. Baseline urine cyclic AMP/plasma AVP ratio distributed between 0.25 and 4.01 with median 1.90. The urine cyclic AMP/plasma AVP ratio was a significant predictor of response to tolvaptan, which was adjusted for 6 potential confounders with a cutoff of 1.24. CONCLUSIONS: Baseline urine cyclic AMP/plasma AVP ratio is an independent predictor of response to tolvaptan in advanced CKD patients with heart failure. CLINICAL TRIAL REGISTRATION: UMIN000022422.
Assuntos
Arginina Vasopressina , AMP Cíclico , Insuficiência Cardíaca , Insuficiência Renal Crônica , Tolvaptan , Idoso , Humanos , Masculino , Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Arginina Vasopressina/sangue , Arginina Vasopressina/química , Benzazepinas/uso terapêutico , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológico , Estudos Prospectivos , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/tratamento farmacológico , Tolvaptan/uso terapêutico , AMP Cíclico/química , AMP Cíclico/urinaRESUMO
CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.
Assuntos
Bactérias , Bacteriófagos , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Nucleotídeos Cíclicos , Protease La , Bactérias/enzimologia , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Óperon , Protease La/química , Protease La/metabolismo , RNA Viral , Fator sigma , Transcrição GênicaRESUMO
Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of Mycobacterium tuberculosis DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after M. tuberculosis infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After M. tuberculosis infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after M. tuberculosis infection. It was found that rBCG-DisA resided longer than BCG in the lung of M. tuberculosis infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against M. tuberculosis infection. Our findings provided in vivo experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What's more, rBCG-DisA showed promising potential in prime-boost strategy against M. tuberculosis infection in adults.
Assuntos
AMP Cíclico , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Aciltransferases/genética , Monofosfato de Adenosina , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Animais , Antígenos de Bactérias , Vacina BCG , AMP Cíclico/química , Citocinas/metabolismo , Fosfatos de Dinucleosídeos , Camundongos , Camundongos Endogâmicos C57BL , Vacinas SintéticasRESUMO
Genetically encoded FRET sensors for revealing local concentrations of second messengers in living cells have enormously contributed to our understanding of physiological and pathological processes. However, the development of sensors remains an intricate process. Using simulation techniques, we recently introduced a new architecture to measure intracellular concentrations of cAMP named CUTie, which works as a FRET tag for arbitrary targeting domains. Although our method showed quasi-quantitative predictive power in the design of cAMP and cGMP sensors, it remains intricate and requires specific computational skills. Here, we provide a simplified computer-aided protocol to design tailor-made CUTie sensors based on arbitrary cyclic nucleotide-binding domains. As a proof of concept, we applied this method to construct a new CUTie sensor with a significantly higher cAMP sensitivity (EC50 = 460 nM).This simple protocol, which integrates our previous experience, only requires free web servers and can be straightforwardly used to create cAMP sensors adapted to the physicochemical characteristics of known cyclic nucleotide-binding domains.
Assuntos
AMP Cíclico , Pedestres , AMP Cíclico/química , GMP Cíclico , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Sistemas do Segundo MensageiroRESUMO
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
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
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
Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.
Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/química , AMP Cíclico/química , Mutação , Subunidades Proteicas/química , Regulação Alostérica , Animais , Sítios de Ligação , Complexo de Carney/enzimologia , Complexo de Carney/genética , Complexo de Carney/patologia , Bovinos , Cristalografia por Raios X , AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Disostoses/enzimologia , Disostoses/genética , Disostoses/patologia , Ativação Enzimática , Expressão Gênica , Humanos , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Cinética , Modelos Moleculares , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.
Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Potássio/metabolismo , Bacillus subtilis , Sítios de Ligação , AMP Cíclico/química , Homeostase , Antiportadores de Potássio-Hidrogênio/química , Ligação ProteicaRESUMO
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
Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.
Assuntos
Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Mutantes/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzotiazóis/química , Benzotiazóis/metabolismo , Sítios de Ligação , AMP Cíclico/química , AMP Cíclico/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Ligantes , Domínios Proteicos , Homologia Estrutural de Proteína , Termodinâmica , Triazóis/química , Triazóis/metabolismoRESUMO
Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRPMTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRPMTB remain unclear. Here, we investigate the binding mechanism between CRPMTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRPMTB forms high-order CRPMTB-DNA oligomers through interactions with nonspecific DNA sequences or preformed CRPMTB-DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRPMTB-DNA oligomers, reduces the affinity of CRPMTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRPMTB-DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRPEcoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRPMTB-DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRPBCG). The functional behavior, thermodynamic stability, and dimerization constant of CRPBCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRPEcoli, illustrating that structural homology does not imply allosteric homology.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Mycobacterium tuberculosis/metabolismo , Regulação Alostérica/fisiologia , Sítios de Ligação , AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , DNA/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Conformação Proteica , Transdução de Sinais , TermodinâmicaRESUMO
Conversion of adenosine triphosphate (ATP) to the second messenger cyclic adenosine monophosphate (cAMP) is an essential reaction mechanism that takes place in eukaryotes, triggering a variety of signal transduction pathways. ATP conversion is catalyzed by the enzyme adenylyl cyclase (AC), which can be regulated by binding inhibitory, Gαi, and stimulatory, Gαs subunits. In the past twenty years, several crystal structures of AC in isolated form and complexed to Gαs subunits have been resolved. Nevertheless, the molecular basis of the inhibition mechanism of AC, induced by Gαi, is still far from being fully understood. Here, classical molecular dynamics simulations of the isolated holo AC protein type 5 and the holo binary complex AC5:Gαi have been analyzed to investigate the conformational impact of Gαi association on ATP-bound AC5. The results show that Gαi appears to inhibit the activity of AC5 by preventing the formation of a reactive ATP conformation.
Assuntos
Adenilil Ciclases/química , AMP Cíclico/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Humanos , Estrutura Quaternária de ProteínaRESUMO
CBP [cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein] is one of the most researched proteins for its therapeutic function. Several studies have identified its vast functions and interactions with other transcription factors to initiate cellular signals of survival. In cancer and other diseases such as Alzheimer's, Rubinstein-taybi syndrome, and inflammatory diseases, CBP has been implicated and hence an attractive target in drug design and development. In this review, we explore the various computational techniques that have been used in CBP research, furthermore we identified computational gaps that could be explored to facilitate the development of highly therapeutic CBP inhibitors.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antineoplásicos/uso terapêutico , Proteína de Ligação a CREB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Síndrome de Rubinstein-Taybi/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Antineoplásicos/química , Sítios de Ligação , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fármacos Neuroprotetores/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Elementos de Resposta , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/metabolismo , Síndrome de Rubinstein-Taybi/patologiaRESUMO
Interaction of follicle stimulating hormone (FSH) with its cognate receptor (FSHR) is critical for maintaining reproductive health. FSHR has a large extracellular domain (ECD), composed of leucine rich repeats (LRRs) and hinge region, a transmembrane domain (TMD) and a short C-terminal domain (CTD). In this study, we have identified a short peptidic stretch in the hinge region (hFSHR(271-275)), through extensive computational modeling, docking and MD simulations, that is capable of independently interacting with the extracellular loops of FSHR(TMD). In vitro studies revealed that FSHR(271-275) peptide increased binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. Administration of FSHR(271-275) peptide in immature female rats significantly increased FSH-mediated ovarian weight gain and promoted granulosa cell proliferation. In summary, the results demonstrate that the synthetic peptide corresponding to amino acids 271-275 of hFSHR-hinge region stimulates FSH-FSHR interaction and behaves as positive allosteric modulator of FSHR. The study also lends evidence to the existing proposition that hinge region maintains the receptor in an inactive conformation in the absence of its ligand by engaging in intramolecular interactions with extracellular loops of TMD.
Assuntos
Células da Granulosa/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos , Receptores do FSH/química , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/química , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Cyclic adenosine monophosphate (cAMP) is a second messenger involved in the dental regeneration. However, efficient long-lasting delivery of cAMP that is sufficient to mimic the in vivo microenvironment remains a major challenge. Here, cAMP was loaded in stem cells from apical papilla (SCAPs) using layer-by-layer self-assembly with gelatin and alginate polyelectrolytes (LBL-cAMP-SCAPs). LBL-cAMP-SCAPs expressed cAMP and increased the phosphorylation level of cAMP-response element-binding protein (CREB) which were evaluated by immunofluorescence and western blotting (WB). Enzyme-linked immunosorbent assay (ELISA) demonstrated that a sustained release of cAMP and vascular endothelial growth factor (VEGF) were present up to 14 days. Scanning electron microscopy (SEM) found LBL-coated SCAPs exhibited a spheroid-like morphology. CCK8 and live/dead staining showed that LBL treatment had no significant effect on cell proliferation and viability. LBL-cAMP-SCAPs enhanced mineralized nodule formation and up-regulated the mRNA levels of the osteogenesis-related genes, as well as related transcription factor-2 protein level which were revealed by Alizarin red staining, RT-PCR and WB, respectively. In conclusion, LBL self-assembly loaded with cAMP promoted the osteo/odontogenic differentiation of SCAPs, thereby providing a potential strategy for bioactive molecular delivery in dental regeneration.
Assuntos
AMP Cíclico/química , Papila Dentária/efeitos dos fármacos , Odontogênese/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Polieletrólitos/química , Células-Tronco/efeitos dos fármacos , Alginatos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Papila Dentária/citologia , Gelatina/química , Humanos , Odontogênese/genética , Osteogênese/genética , RNA Mensageiro/biossíntese , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Small molecules that bind the SARS-CoV-2 nonstructural protein 3 Mac1 domain in place of ADP-ribose could be useful as molecular probes or scaffolds for COVID-19 antiviral drug discovery because Mac1 has been linked to the ability of coronaviruses to evade cellular detection. A high-throughput assay based on differential scanning fluorimetry (DSF) was therefore optimized and used to identify possible Mac1 ligands in small libraries of drugs and drug-like compounds. Numerous promising compounds included nucleotides, steroids, ß-lactams, and benzimidazoles. The main drawback to this approach was that a high percentage of compounds in some libraries were found to influence the observed Mac1 melting temperature. To prioritize DSF screening hits, the shapes of the observed melting curves and initial assay fluorescence were examined, and the results were compared with virtual screens performed using AutoDock Vina. The molecular basis for alternate ligand binding was also examined by determining a structure of one of the hits, cyclic adenosine monophosphate, with atomic resolution.