RESUMO
P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.
Assuntos
Trifosfato de Adenosina , Microscopia Crioeletrônica , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X1 , Humanos , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X1/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Ligantes , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ligação Proteica , Sítios de Ligação , Células HEK293 , Modelos Moleculares , BenzenossulfonatosRESUMO
In this study, a turn-on fluorescence sensor for the detection of adenosine 5'-triphosphate (ATP) was developed and tested using ATP-DNA2-Ag NCs. The results showed that the fluorescence of ATP-DNA2-Ag NCs was significantly enhanced with the addition of ATP. The fluorescence enhancement was a result of the specific binding activity of the ATP aptamer and ATP, which caused G-rich sequences to approach the dark DNA-Ag NCs, owing to the alteration in ATP aptamer conformation. The proposed sensor demonstrated a good linear range of 18-42 mM and a limit of detection (LOD) of 2.8 µM. The sensor's features include sensitivity, selectivity, and simple operation. In addition, the proposed sensor successfully measured ATP in 100-fold diluted fetal bovine serum.
Assuntos
Trifosfato de Adenosina , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA , Nanopartículas Metálicas , Prata , Espectrometria de Fluorescência , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Prata/química , Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , DNA/química , Aptâmeros de Nucleotídeos/química , Espectrometria de Fluorescência/métodos , Limite de Detecção , Fluorescência , Animais , BovinosRESUMO
Tumor-associated antigens (TAAs) are not exclusively expressed in cancer cells, inevitably causing the "on target, off tumor" effect of molecular recognition tools. To achieve precise recognition of cancer cells, by using protein tyrosine kinase 7 (PTK7) as a model TAA, a DNA molecular logic circuit Aisgc8 was rationally developed by arranging H+-binding i-motif, ATP-binding aptamer, and PTK7-targeting aptamer Sgc8c in a DNA sequence. Aisgc8 output the conformation of Sgc8c to recognize PTK7 on cells in a simulated tumor microenvironment characterized by weak acidity and abundant ATP, but not in a simulated physiological environment. Through in vitro and in vivo results, Aisgc8 demonstrated its ability to precisely recognize cancer cells and, as a result, displayed excellent performance in tumor imaging. Thus, our studies produced a simple and efficient strategy to construct DNA logic circuits, opening new possibilities to develop convenient and intelligent precision diagnostics by using DNA logic circuits.
Assuntos
Aptâmeros de Nucleotídeos , Humanos , Aptâmeros de Nucleotídeos/química , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , Receptores Proteína Tirosina Quinases/genética , Linhagem Celular Tumoral , Antígenos de Neoplasias/genética , Computadores Moleculares , Animais , DNA/química , Microambiente Tumoral , Camundongos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Moléculas de Adesão CelularRESUMO
Temporal control over self-assembly processes is a highly desirable attribute that is efficiently exhibited by biological systems, such as actin filaments. In nature, various proteins undergo enzymatically catalysed chemical reactions that kinetically govern their structural and functional properties. Consequently, any stimuli that can alter their reaction kinetics can lead to a change in their growth or decay profiles. This underscores the urgent need to investigate bioinspired, adaptable and controllable synthetic materials. Herein we intend to develop a general strategy for controlling the growth and decay of self-assembled systems via enzymatically coupled reactions. We achieve this by the coupling of enzymes phosphokinase/phosphatase with a bolaamphiphilic cationic chromophore (PDI) which selectively self-assembles with ATP and disassembles upon its enzymatic hydrolysis. The aggregation process is efficiently regulated by the controlled in situ generation of ATP, through enzymatic reactions. By carefully managing the ATP generating components, we realize precise control over the self-assembly process. Moreover, we also show self-assembled structures with programmed temporal decay profiles through coupled enzymatic reactions of ATP generation and hydrolysis, essentially rendering the process dissipative. This work introduces a novel strategy to generate a reaction-coupled one-dimensional nanostructure with controlled dimensions inspired by biological systems.
Assuntos
Trifosfato de Adenosina , Biocatálise , Polimerização , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Hidrólise , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Estrutura MolecularRESUMO
Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.
Assuntos
Domínio Catalítico , Magnésio , Simulação de Dinâmica Molecular , RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/química , RNA Polimerase II/genética , Magnésio/metabolismo , Magnésio/química , Lasers , Conformação Proteica , Elétrons , Ligação Proteica , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Sítios de LigaçãoRESUMO
ConspectusIn recent years, the controlled assembly/disassembly of exogenous chemical components inside cells has become an emerging approach to regulating cell functions. However, the construction of dynamic material chemistry systems in living cells always remains highly challenging due to the complicated intracellular microenvironment. Nucleic acid is a category of biological components that can achieve efficient molecular assembly via specific base-pairing and perform biological functions in the intracellular microenvironment. Deoxyribonucleic acid (DNA) molecules exhibit the superior performance of intracellular assembly, including sequence programmability, molecule recognition ability, and nanostructure predictability, as well as the unique biological functions that traditional synthetic polymers do not carry, showing great superiority in the construction of dynamic material chemistry systems. Moreover, the technologies of DNA synthesis are relatively mature, and the conjugation of DNA with functional small molecules can be achieved through established chemical synthesis methods, facilitating the construction of DNA-based dynamic materials with more functions. In addition, a few specific DNA molecules have been proven to show responsiveness toward different stimuli, functioning as dynamic modules.In this Account, we summarize our recent work in dynamic chemistry of DNA-based nanoassemblies in living cells from the perspective of stimulus types including enzyme, H+, glutathione (GSH), adenosine triphosphate (ATP), and light. Upon the specific stimuli, DNA-based nanoassemblies undergo precise assembly in living cells, executing disassembly or aggregation, which consequently affects the functions and behaviors of living cells. In the first part, we describe the interactions between DNA-based nanoassemblies and intracellular enzymes, namely the enzymatic cleavage of intracellular enzymes on the DNA or RNA sequences. In the second part, we summarize the effects of H+ in lysosomes on DNA-based nanoassemblies, including the formation of a tetraplex i-motif structure and the decomposition of acid-degradable polymeric coating. In the third part, we discuss the mechanism of GSH responsiveness of DNA-based nanoassemblies, including the breaking of disulfide bonds and reduction-responsive nanoparticles. In the fourth part, we describe the ATP-mediated conformational transition for the specific release of functional RNA sequences. In the fifth part, we demonstrate the light-mediated spatiotemporally dynamic chemistry of DNA-based nanoassemblies. In summary, based on the achievements of our group in the study of dynamic chemistry of DNA-based nanoassemblies, the assembly, disassembly, and reassembly in living cells are well-controlled, the regulation of cellular functions are explored, and the new strategies for cancer therapeutics are demonstrated. We envision that our work on the dynamic chemistry of DNA-based nanoassembly is a new paradigm for constructing dynamic material chemistry systems inside living cells, and will facilitate the development of precision medicine.
Assuntos
DNA , Nanoestruturas , DNA/química , DNA/metabolismo , Humanos , Nanoestruturas/química , Glutationa/química , Glutationa/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , LuzRESUMO
The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.
Assuntos
Domínio Catalítico , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Mitocôndrias , Humanos , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , Especificidade por Substrato , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Ligação Proteica , Sítios de LigaçãoRESUMO
Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.
Assuntos
Trifosfato de Adenosina , Aptâmeros de Nucleotídeos , Carbono , Nucleolina , Pontos Quânticos , Pontos Quânticos/química , Aptâmeros de Nucleotídeos/química , Humanos , Carbono/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/análise , Corantes Fluorescentes/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , DNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Células HeLa , Imagem Óptica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/química , Moléculas de Adesão Celular , Receptores Proteína Tirosina QuinasesRESUMO
Reconfiguration of chemical sensors, intended as the capacity of the sensor to adapt to novel operational scenarios, e.g., new target analytes, is potentially game changing and would enable rapid and cost-effective reaction to dynamic changes occurring at healthcare, environmental, and industrial levels. Yet, it is still a challenge, and rare examples of sensor reconfiguration have been reported to date. Here, we report on a reconfigurable label-free optical sensor leveraging the versatile immobilization of metal ions through a chelating agent on a nanostructured porous silica (PSiO2) optical transducer for the detection of different biomolecules. First, we show the reversible grafting of different metal ions on the PSiO2 surface, namely, Ni2+, Cu2+, Zn2+, and Fe3+, which can mediate the interaction with different biomolecules and be switched under mild conditions. Then, we demonstrate reconfiguration of the sensor at two levels: 1) switching of the metal ions on the PSiO2 surface from Cu2+ to Zn2+ and testing the ability of Cu2+-functionalized and Zn2+-reconfigured devices for the sensing of the dipeptide carnosine (CAR), leveraging the well-known chelating ability of CAR toward divalent metal ions; and 2) reconfiguration of the Cu2+-functionalized PSiO2 sensor for a different target analyte, namely, the nucleotide adenosine triphosphate (ATP), switching Cu2+ with Fe3+ ions to exploit the interaction with ATP through phosphate groups. The Cu2+-functionalized and Zn2+-reconfigured sensors show effective sensing performance in CAR detection, also evaluated in tissue samples from murine brain, and so does the Fe3+-reconfigured sensor toward ATP, thus demonstrating effective reconfiguration of the sensor with the proposed surface chemistry.
Assuntos
Técnicas Biossensoriais , Dióxido de Silício , Animais , Camundongos , Dióxido de Silício/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Nanoestruturas/química , Porosidade , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Cobre/química , Metais/químicaRESUMO
Mitogen-activated protein kinase kinases (MAP2Ks) 1, 4, and 7 are potential targets for treating various diseases. Here, we solved the crystal structures of MAP2K1 and MAP2K4 complexed with covalent inhibitor 5Z-7-oxozeaenol (5Z7O). The elucidated structures showed that 5Z7O was non-covalently bound to the ATP binding site of MAP2K4, while it covalently attached to cysteine at the DFG-1 position of the deep ATP site of MAP2K1. In contrast, we previously showed that 5Z7O covalently binds to MAP2K7 via another cysteine on the solvent-accessible edge of the ATP site. Structural analyses and molecular dynamics calculations indicated that the configuration and mobility of conserved gatekeeper methionine located at the central ATP site regulated the binding and access of 5Z7O to the ATP site of MAP2Ks. These structural features provide clues for developing highly potent and selective inhibitors against MAP2Ks. Abbreviations: ATP, adenosine triphosphate; FDA, Food and Drug Administration; MAP2Ks, mitogen-activated protein kinase kinases; MD, molecular dynamics; NSCLC, non-small cell lung cancer; 5Z7O, 5Z-7-oxozeaenol; PDB, protein data bank; RMSD, root-mean-square deviation.
Assuntos
Trifosfato de Adenosina , Metionina , Inibidores de Proteínas Quinases , Zearalenona , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Humanos , Metionina/química , Metionina/metabolismo , Sítios de Ligação , Zearalenona/análogos & derivados , Zearalenona/química , Zearalenona/farmacologia , Zearalenona/metabolismo , Zearalenona/administração & dosagem , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/química , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 7/metabolismo , MAP Quinase Quinase 7/antagonistas & inibidores , MAP Quinase Quinase 7/química , Relação Estrutura-Atividade , Simulação de Dinâmica Molecular , Cristalografia por Raios X , Estrutura Molecular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Lactonas , Resorcinóis , MAP Quinase Quinase 4RESUMO
Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90ß chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90ß inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90ß inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90ß linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90ß. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.
Assuntos
Benzoquinonas , Proteínas de Choque Térmico HSP90 , Lactamas Macrocíclicas , Ritonavir , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/química , Ritonavir/química , Ritonavir/farmacologia , Benzoquinonas/química , Benzoquinonas/farmacologia , Benzoquinonas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Ligação Proteica , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Modelos Moleculares , Sítios de Ligação , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/químicaRESUMO
Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.
Assuntos
Adenilato Quinase , Domínio Catalítico , Magnésio , Simulação de Dinâmica Molecular , Magnésio/metabolismo , Magnésio/química , Adenilato Quinase/metabolismo , Adenilato Quinase/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Conformação Proteica , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/químicaRESUMO
Although cellular transport machinery is mostly ATP-driven and ATPase-dependent, there has been a recent surge in understanding colloidal transport processes relying on a nonspecific physical interaction with biologically significant small molecules. Herein, we probe the phoretic behavior of a biocolloid [composed of a Zn(II)-coordinated metallomicelle and enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx)] when exposed to a concentration gradient of ATP under microfluidic conditions. Simultaneously, we demonstrate that an ATP-independent oxidative biocatalytic product formation zone can be modulated in the presence of a (glucose + ATP) gradient. We report that both directionality and extent of transport can be tuned by changing the concentration of the ATP gradient. This diffusiophoretic mobility of a submicrometer biocolloidal object for the spatial transposition of a biocatalytic zone signifies the ATP-mediated functional transportation without the involvement of ATPase. Additionally, the ability to analyze colloidal transport in microfluidic channels using an enzymatic fluorescent product-forming reaction could be a new nanobiotechnological tool for understanding transport and spatial catalytic patterning processes. We believe that this result will inspire further studies for the realization of elusive biological transport processes and target-specific delivery vehicles, considering the omnipresence of the ATP-gradient across the cell.
Assuntos
Trifosfato de Adenosina , Biocatálise , Glucose Oxidase , Peroxidase do Rábano Silvestre , Zinco , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Zinco/química , Zinco/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Oxirredução , Coloides/químicaRESUMO
Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.
Assuntos
Difosfato de Adenosina , Trifosfato de Adenosina , Proteínas de Choque Térmico HSP70 , Simulação de Dinâmica Molecular , Mutação , Humanos , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/química , Conformação ProteicaRESUMO
GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroELMEL, in which the 307â309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroELMEL-mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent.
Assuntos
Trifosfato de Adenosina , Antracenos , Chaperonina 60 , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Hidrólise , Antracenos/química , Antracenos/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Mutação , Ligação de Hidrogênio , Dobramento de Proteína , Ácidos CarboxílicosRESUMO
The simple (self-)coacervation of the minimal tryptophan/arginine peptide sequences W2R2 and W3R3 was observed in salt-free aqueous solution. The phase diagrams were mapped using turbidimetry and optical microscopy, and the coacervate droplets were imaged using confocal microscopy complemented by cryo-TEM to image smaller droplets. The droplet size distribution and stability were probed using dynamic light scattering, and the droplet surface potential was obtained from zeta potential measurements. SAXS was used to elucidate the structure within the coacervate droplets, and circular dichroism spectroscopy was used to probe the conformation of the peptides, a characteristic signature for cation-π interactions being present under conditions of coacervation. These observations were rationalized using a simple model for the Rayleigh stability of charged coacervate droplets, along with atomistic molecular dynamics simulations which provide insight into stabilizing π-π stacking interactions of tryptophan as well as arginine-tryptophan cation-π interactions (which modulate the charge of the tryptophan π-electron system). Remarkably, the dipeptide WR did not show simple coacervation under the conditions examined, but complex coacervation was observed in mixtures with ATP (adenosine triphosphate). The electrostatically stabilized coacervation in this case provides a minimal model for peptide/nucleotide membraneless organelle formation. These are among the simplest model peptide systems observed to date able to undergo either simple or complex coacervation and are of future interest as protocell systems.
Assuntos
Trifosfato de Adenosina , Trifosfato de Adenosina/química , Triptofano/química , Simulação de Dinâmica Molecular , Peptídeos/química , Arginina/química , Separação de FasesRESUMO
This study reports a fluorescent nanoprobe operated in fluorescence turn-on mode for simultaneously sensing and imaging intracellular GSH and ATP. By using maleimide-derivatives as the ligand, the bimetallic nanoscale metal-organic framework (NMOF) Cu-Mi-UiO-66 has been synthesized for the first time using a straightforward one-step solvothermal approach, serving as a GSH recognition moiety. Subsequently, a Cy5-labeled ATP aptamer was assembled onto Cu-Mi-UiO-66 via strong coordination between phosphate and zirconium, π-π stacking and electrostatic adsorption to develop the dual-responsive fluorescence nanoprobe Cu-Mi-UiO-66/aptamer. Due to the photoinduced electron transfer (PET) effect between maleimide groups and the benzene ring of the ligand and the charge transfer between Cy5 and the Zr(IV)/Cu(II) bimetal center of the NMOF, the Cu-Mi-UiO-66/aptamer exhibits a fluorescence turn-off status. The Michael addition reaction between the thiol group of GSH and the maleimide on the NMOF skeleton results in turning on of the blue fluorescence of Cu-Mi-UiO-66. Meanwhile, upon specific interaction with ATP, the aptamer changes into internal loop structures and detaches from Cu-Mi-UiO-66, resulting in turning on of the red fluorescence of Cy5. The nanoprobe demonstrated an excellent sensing performance with a good linear range (GSH, 5.0-450.0 µM; ATP, 1.0-50.0 µM) and a low detection limit (GSH, 2.17 µM; ATP, 0.635 µM). More importantly, the Cu-Mi-UiO-66/aptamer exhibits good performance for tracing intracellular concentration variations of GSH and ATP in living HepG2 cells under different stimulations. This study highlights the potential of NMOFs for multiplexed analysis and provides a valuable tool for tumor microenvironment research and early cancer diagnosis.
Assuntos
Trifosfato de Adenosina , Cobre , Corantes Fluorescentes , Glutationa , Estruturas Metalorgânicas , Glutationa/análise , Glutationa/química , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Humanos , Corantes Fluorescentes/química , Cobre/química , Estruturas Metalorgânicas/química , Aptâmeros de Nucleotídeos/química , Zircônio/química , Carbocianinas/química , Espectrometria de Fluorescência , Ácidos FtálicosRESUMO
The F1-ATPase enzyme is the smallest-known molecular motor that rotates in 120° steps, driven by the hydrolysis of ATP. It is a multi-subunit enzyme that contains three catalytic sites. A central question is how the elementary chemical reactions that occur in the three sites are coupled to mechanical rotation. Various models and coupling schemes have been formulated in an attempt to answer this question. They can be classified as 2-site (bi-site) models, exemplified by Boyer's binding change mechanism first proposed 50 years ago, and 3-site (tri-site) models such as Nath's torsional mechanism, first postulated 25 years ago and embellished 1 year back. Experimental data collated using diverse approaches have conclusively shown that steady-state ATP hydrolysis by F1-ATPase occurs in tri-site mode. Hence older models have been continually modified to make them conform to the new facts. Here, we have developed a pure mathematical approach based on combinatorics and conservation laws to test if proposed models are 2-site or 3-site. Based on this novel combinatorial approach, we have proved that older and modified models are effectively biâsite models in that catalysis and rotation in F1-ATPase occurs in these models with only two catalytic sites occupied by bound nucleotide. Hence these models contradict consensus experimental data. The recent 2023 model of ATP hydrolysis by F1-ATPase has been proved to be a true tri-site model based on our novel mathematical approach. Such pure mathematical proofs constitute an important step forward for ATP mechanism. However, in what must be considered an aspect with great scientific potential, the power of such mathematical proofs has not been fully exploited to solve molecular biological problems, in our opinion. We believe that the creative application of pure mathematical proofs (for another example see Nath in Theory Biosci 141:249-260, 2022) can help resolve with finality various longstanding molecular-level issues that arise as a matter of course in the analysis of fundamental biological problems. Such issues have proved extraordinarily difficult to resolve by standard experimental, theoretical, or computational approaches.
Assuntos
Trifosfato de Adenosina , ATPases Translocadoras de Prótons , Hidrólise , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Domínio Catalítico , Cinética , Algoritmos , Catálise , Rotação , Sítios de Ligação , Modelos MolecularesRESUMO
The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.
Assuntos
Trifosfato de Adenosina , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Estabilidade Proteica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/química , Ligação Proteica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/química , Domínios Proteicos , Magnésio/metabolismo , Magnésio/químicaRESUMO
Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multispecificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.