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1.
Life Sci ; 248: 117477, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119961

RESUMO

AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Guanosina Monofosfato/análogos & derivados , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virais/antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimologia , Alphacoronavirus/genética , Sequência de Aminoácidos , Antivirais/metabolismo , Betacoronavirus/genética , Domínio Catalítico , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Replicase/química , RNA Replicase/metabolismo , Ribavirina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sofosbuvir/metabolismo , Termodinâmica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
2.
J Chem Theory Comput ; 15(12): 6992-7003, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31714772

RESUMO

Di/triphosphates perform a multitude of essential tasks, being important components of many vital organic cofactors such as adenosine/guanosine di/triphosphate (ADP/GDP, ATP/GTP), flavin adenine dinucleotide, and nicotinamide adenine dinucleotide and its phosphate derivative. They are generally bound to cations inside cells, in particular Mg2+ in the case of ATP/GTP. Yet how their metal-binding modes depend on the number, charge, and solvent exposure of the polyphosphate group and how Mg2+and Ca2+ dications that coexist in cellular fluids compete for di/triphosphates in biological systems remain elusive. Using density functional theory calculations combined with a polarizable continuum model, we have determined the relative free energies and stabilities of the different binding modes of di- and triphosphate groups to Mg2+ and Ca2+. We show that the thermodynamic outcome of the competition between Mg2+ and Ca2+ for cellular di/triphosphates depends mainly on the oligomericity/charge and metal-binding mode of the phosphate ligand as well as the solvent exposure of the binding site. Increasing the charge and thus denticity of the phosphate ligand from bi- to tridentate in a buried binding pocket enhances the affinity of the host system for the stronger charge acceptor, Mg2+. The cellular di/triphosphates's intrinsic properties and the protein matrix allowing them to bind a dication bi/tridentately, along with the higher cytosolic concentration of Mg2+ compared to Ca2+, enables Mg2+ to outcompete Ca2+ in binding to these highly charged anions. This suggests an explanation for why nature has chosen Mg2+ but not Ca2+ to perform most of the essential tasks associated with biological triphosphates.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Cálcio , Guanosina Difosfato/química , Guanosina Trifosfato/química , Magnésio/química , Sítios de Ligação , Cálcio/química , Bases de Dados de Proteínas , Teoria da Densidade Funcional , Termodinâmica
3.
Life Sci ; 238: 116958, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31628915

RESUMO

AIM: To work on Hepatitis C Virus (HCV), one of the major causes of liver cirrhosis and hepatocellular carcinoma, polymerase of genotype 4a that have no solved structures deposited in the protein data bank (PDB) yet. Understanding the dynamics and testing some novel inhibitors are also covered. MATERIALS AND METHODS: Molecular Dynamics Simulation (MDS) is performed for a period of 1 µs on comparatively modeled then validated NS5b of subtype 4a. Following MDS analysis, molecular docking is performed to test the inhibitory performance of eight novels suggested guanosine derivatives using 181 different conformations of the protein model gathered during the MDS run after the equilibration period. KEY FINDINGS: The results yield that the eight modified, at position 2', GTP derivatives (fluorine, Hydroxyl, and sulphonyl oxydanyl) have binding energies comparable to the parent molecule, GTP. Besides, the eight suggested compounds have lower binding energies (and hence better in binding) compared to sofosbuvir (a drug approved by FDA in 2013 against HCV) and ribavirin (a wide range acting antiviral drug used before against HCV). SIGNIFICANCE: Combined molecular dynamics and molecular docking are able to test the hypothesis of HCV polymerase dynamics doesn't affect the nucleotides (or nucleotide inhibitors) binding to its active site. Despite the reported highly dynamic subtype 4a of HCV; all the nucleotide inhibitors under the study are able to, tightly, bind to NS5b of genotype 4a. This behavior is reported before for the Zika virus polymerase, as well.


Assuntos
Antivirais/química , Antivirais/farmacologia , Guanosina Trifosfato/química , Hepacivirus/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas não Estruturais Virais/antagonistas & inibidores , Hepacivirus/classificação , Hepacivirus/genética , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
4.
Molecules ; 24(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614753

RESUMO

Starch digestibility may be affected by food microstructural changes, as well as by specific interactions with some biomolecules, such as soluble dietary fibers (SDFs). It is well-known that acrylamide (AA) is a toxic and potentially carcinogenic compound formed in starchy food products processed at temperatures above 120 °C. This study aimed to investigate the effect of the addition of SDF and green tea polyphenols (GTP) on AA formation and in vitro starch digestibility in baked starchy matrices. The formulations were prepared using gluten and wheat starch, ensuring ~40 ± 2% (wet basis, w.b.) moisture in the doughs. In some samples, 7.5% (dry basis, d.b.) of starch was replaced with inulin (IN), polydextrose (PD) or partially hydrolyzed guar gum (PHGG), and/or with GTP at 1% (d.b). Acrylamide was determined by gas chromatography-mass spectrometry, and the in vitro starch digestibility using the Englyst method. The GTP was able to reduce AA content by ~48%, and a combination of IN-GTP allowed it to be reduced by up to ~64%, revealing the lowest rapidly available glucose content (~17 mg/g glucose). While a PD-GTP mixture reduced the AA content by around ~57% and gave the highest unavailable glucose fraction (~74 mg/g glucose) compared to the control. This study showed how functional ingredients could be used to develop successfully healthier starchy bakery foods.


Assuntos
Fibras na Dieta , Digestão/efeitos dos fármacos , Polifenóis/farmacologia , Amido/química , Acrilamida/química , Glucose/química , Guanosina Trifosfato/química , Humanos , Polifenóis/química , Solubilidade/efeitos dos fármacos , Amido/metabolismo , Chá/química
5.
Nat Struct Mol Biol ; 26(11): 1013-1022, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659330

RESUMO

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.


Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/química , Guanosina Trifosfato/química , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Forma A/química , DNA de Forma B/química , Modelos Moleculares , Conformação Proteica
7.
Soft Matter ; 15(24): 4797-4807, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31123741

RESUMO

Microtubule self-organization is an essential physical process underlying several essential cellular functions, including cell division. In cell division, the dominant arrangement is the mitotic spindle, a football-shaped microtubule-based machine responsible for separating the chromosomes. We are interested in the underlying fundamental principles behind the self-organization of the spindle shape. Prior biological works have hypothesized that motor proteins control the proper formation of the spindle. Many of these motor proteins are also microtubule-crosslinkers, so it is unclear if the critical aspect is the motor activity or the crosslinking. In this study, we seek to address this question by examining the self-organization of microtubules using crosslinkers alone. We use a minimal system composed of tubulin, an antiparallel microtubule-crosslinking protein, and a crowding agent to explore the phase space of organizations as a function of tubulin and crosslinker concentration. We find that the concentration of the antiparallel crosslinker, MAP65, has a significant effect on the organization and resulted in spindle-like arrangements at relatively low concentration without the need for motor activity. Surprisingly, the length of the microtubules only moderately affects the equilibrium phase. We characterize both the shape and dynamics of these spindle-like organizations. We find that they are birefringent homogeneous tactoids. The microtubules have slow mobility, but the crosslinkers have fast mobility within the tactoids. These structures represent a first step in the recapitulation of self-organized spindles of microtubules that can be used as initial structures for further biophysical and active matter studies relevant to the biological process of cell division.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Reagentes para Ligações Cruzadas/química , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanosina Trifosfato/química , Análise de Séries Temporais Interrompida , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Paclitaxel/química , Polimerização , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
8.
Biochem J ; 476(10): 1433-1444, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31036719

RESUMO

The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.


Assuntos
Proteínas de Bactérias , Proteínas do Citoesqueleto , Guanosina Trifosfato , Multimerização Proteica , Streptococcus pneumoniae , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólise , Domínios Proteicos , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismo
9.
Org Biomol Chem ; 17(19): 4879-4891, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31041977

RESUMO

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.


Assuntos
Biocatálise , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Proteínas Ativadoras de ras GTPase/metabolismo , Sítios de Ligação , Guanosina Trifosfato/química , Hidrólise , Conformação Molecular , Mutação , Teoria Quântica , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética
10.
Chemistry ; 25(36): 8484-8488, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31038818

RESUMO

Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (Pi ). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA-GAP-GDP-Pi . Its 1.75 Šcrystal structure shows geometry for ionic and hydrogen bond coordination of GDP and Pi in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H-bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate Pi release.


Assuntos
Teoria da Densidade Funcional , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligações de Hidrogênio , Hidrólise , Simulação de Dinâmica Molecular , Fosfatos/química , Prótons
11.
Int J Mol Sci ; 20(10)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137602

RESUMO

We have used a simple model system to test the prediction that surface attachment strength of filaments presenting a torsion would affect their shape and properties. FtsZ from E. coli containing one cysteine in position 2 was covalently attached to a lipid bilayer containing maleimide lipids either in their head group (to simulate tight attachment) or at the end of a polyethylene glycol molecule attached to the head group (to simulate loose binding). We found that filaments tightly attached grew straight, growing from both ends, until they formed a two-dimensional lattice. Further monomer additions to their sides generated a dense layer of oriented filaments that fully covered the lipid membrane. After this point the surface became unstable and the bilayer detached from the surface. Filaments with a loose binding were initially curved and later evolved into straight thicker bundles that destabilized the membrane after reaching a certain surface density. Previously described theoretical models of FtsZ filament assembly on surfaces that include lateral interactions, spontaneous curvature, torsion, anchoring to the membrane, relative geometry of the surface and the filament 'living-polymer' condition in the presence of guanosine triphosphate (GTP) can offer some clues about the driving forces inducing these filament rearrangements.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Bicamadas Lipídicas/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica
12.
Mol Cancer ; 18(1): 85, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971271

RESUMO

BACKGROUND: Lung cancer patients with KRAS mutation(s) have a poor prognosis due in part to the development of resistance to currently available therapeutic interventions. Development of a new class of anticancer agents that directly targets KRAS may provide a more attractive option for the treatment of KRAS-mutant lung cancer. RESULTS: Here we identified a small molecule KRAS agonist, KRA-533, that binds the GTP/GDP-binding pocket of KRAS. In vitro GDP/GTP exchange assay reveals that KRA-533 activates KRAS by preventing the cleavage of GTP into GDP, leading to the accumulation of GTP-KRAS, an active form of KRAS. Treatment of human lung cancer cells with KRA-533 resulted in increased KRAS activity and suppression of cell growth. Lung cancer cell lines with KRAS mutation were relatively more sensitive to KRA-533 than cell lines without KRAS mutation. Mutating one of the hydrogen-bonds among the KRA-533 binding amino acids in KRAS (mutant K117A) resulted in failure of KRAS to bind KRA-533. KRA-533 had no effect on the activity of K117A mutant KRAS, suggesting that KRA-533 binding to K117 is required for KRA-533 to enhance KRAS activity. Intriguingly, KRA-533-mediated KRAS activation not only promoted apoptosis but also autophagic cell death. In mutant KRAS lung cancer xenografts and genetically engineered mutant KRAS-driven lung cancer models, KRA-533 suppressed malignant growth without significant toxicity to normal tissues. CONCLUSIONS: The development of this KRAS agonist as a new class of anticancer drug offers a potentially effective strategy for the treatment of lung cancer with KRAS mutation and/or mutant KRAS-driven lung cancer.


Assuntos
Antineoplásicos/farmacologia , Autofagia/genética , Benzoatos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Autofagia/efeitos dos fármacos , Benzoatos/química , Sítios de Ligação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Camundongos Transgênicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/agonistas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Biol Chem ; 294(20): 8148-8160, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30940727

RESUMO

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1-3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca2+ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca2+ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca2+ than for Mg2+ Finally, we found that Ca2+ promotes interactions between RGS17 and activated Gα and decreases the Km for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca2+ positively regulates RGS17, which may represent a general mechanism by which increased Ca2+ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca2+ signaling.


Assuntos
Sinalização do Cálcio , Cálcio/química , Proteínas RGS/química , Cálcio/metabolismo , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Magnésio/química , Magnésio/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo
14.
J Biol Chem ; 294(19): 7573-7587, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30867194

RESUMO

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.


Assuntos
Guanosina Trifosfato/química , Hepacivirus/enzimologia , Simulação de Dinâmica Molecular , RNA Replicase/química , Uridina Trifosfato/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Guanosina Trifosfato/metabolismo , RNA Replicase/metabolismo , Uridina Trifosfato/metabolismo , Proteínas não Estruturais Virais/metabolismo
15.
Bioessays ; 41(3): e1800194, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30730055

RESUMO

Microtubules form a highly dynamic filament network in all eukaryotic cells. Individual microtubules grow by tubulin dimer subunit addition and frequently switch between phases of growth and shortening. These unique dynamics are powered by GTP hydrolysis and drive microtubule network remodeling, which is central to eukaryotic cell biology and morphogenesis. Yet, our knowledge of the molecular events at growing microtubule ends remains incomplete. Here, recent ultrastructural, biochemical and cell biological data are integrated to develop a realistic model of growing microtubule ends comprised of structurally distinct but biochemically overlapping zones. Proteins that recognize microtubule lattice conformations associated with specific tubulin guanosine nucleotide states may independently control major structural transitions at growing microtubule ends. A model is proposed in which tubulin dimer addition and subsequent closure of the MT wall are optimized in cells to achieve rapid physiological microtubule growth.


Assuntos
Microtúbulos/metabolismo , Tubulina (Proteína)/química , Animais , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Guanosina/química , Guanosina Trifosfato/química , Humanos , Hidrólise , Mamíferos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Morfogênese , Neuropeptídeos/metabolismo , Polimerização , Ligação Proteica , Conformação Proteica , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/metabolismo
16.
J Biol Chem ; 294(15): 5907-5913, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796162

RESUMO

Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação de Sentido Incorreto , Doença de Parkinson , Multimerização Proteica , Substituição de Aminoácidos , Guanosina Difosfato/química , Guanosina Difosfato/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Domínios Proteicos
17.
J Biol Chem ; 294(8): 2970-2975, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30651352

RESUMO

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex.


Assuntos
Guanosina Trifosfato , Proteínas Monoméricas de Ligação ao GTP , Proteínas Repressoras , Proteínas Supressoras de Tumor , Substituição de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação de Sentido Incorreto , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
18.
Acta Biochim Biophys Sin (Shanghai) ; 51(2): 139-149, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615070

RESUMO

Mycobacterium tuberculosis (Mtb) is the key devastating bacterial pathogen responsible for tuberculosis. Increasing emergence of multi-drug-resistant, extensively drug-resistant, and rifampicin/isoniazid-resistant strains of Mtb makes the discovery of validated drug targets an urgent priority. As a vital translational component of the protein biosynthesis system, elongation factor Tu (EF-Tu) is an important molecular switch responsible for selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. In addition, EF-Tu from Mtb (MtbEF-Tu) is involved in the initial step of trans-translation which is an effective system for rescuing the stalled ribosomes from non-stop translation complexes under stress conditions. Given its crucial role in protein biosynthesis, EF-Tu is identified as an excellent molecular target for drug design. Here, we reported the recombinant expression, purification, biophysical characterization, and structural modeling of the MtbEF-Tu protein. Our results demonstrated that prokaryotic expression plasmids of pET28a-MtbEF-Tu could be expressed efficiently in Escherichia coli. We successfully purified the 6× His-tagged proteins with a yield of 16.8 mg from 1 l of Luria Bertani medium. Dynamic light scattering experiments showed that MtbEF-Tu existed in a monomeric form, and circular dichroism experiments indicated that MtbEF-Tu was well structured. Moreover, isothermal titration calorimetry experiments displayed that the purified MtbEF-Tu protein possessed intermediate binding affinities for guanosine-5'-triphosphate (GTP) and GDP. The GTP/GDP-binding sites were predicted by flexible molecular docking approach which reveals that GTP/GDP binds to MtbEF-Tu mainly through hydrogen bonds. Our work lays the essential basis for further structural and functional studies of MtbEF-Tu as well as MtbEF-Tu-related novel drug developments.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Ligantes , Modelos Moleculares , Mutação , Mycobacterium tuberculosis/genética , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Ligação Proteica , Domínios Proteicos , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo
19.
Biochem J ; 476(1): 51-66, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30538153

RESUMO

The LRR (leucine-rich repeat)-Roc (Ras of complex proteins)-COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer-monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5'-diphosphate)-bound states and monomeric in the GTP (guanosine-5'-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR-Roc-COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the 'Roc dimerization loop'. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.


Assuntos
Proteínas de Bactérias/química , Chlorobium/enzimologia , Dimerização , Guanosina Trifosfato/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Simulação de Dinâmica Molecular , Proteínas de Bactérias/genética , Chlorobium/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Estrutura Terciária de Proteína
20.
Elife ; 72018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30526846

RESUMO

The ubiquitous P-loop fold nucleoside triphosphatases (NTPases) are typically activated by an arginine or lysine 'finger'. Some of the apparently ancestral NTPases are, instead, activated by potassium ions. To clarify the activation mechanism, we combined comparative structure analysis with molecular dynamics (MD) simulations of Mg-ATP and Mg-GTP complexes in water and in the presence of potassium, sodium, or ammonium ions. In all analyzed structures of diverse P-loop NTPases, the conserved P-loop motif keeps the triphosphate chain of bound NTPs (or their analogs) in an extended, catalytically prone conformation, similar to that imposed on NTPs in water by potassium or ammonium ions. MD simulations of potassium-dependent GTPase MnmE showed that linking of alpha- and gamma phosphates by the activating potassium ion led to the rotation of the gamma-phosphate group yielding an almost eclipsed, catalytically productive conformation of the triphosphate chain, which could represent the basic mechanism of hydrolysis by P-loop NTPases.


Assuntos
Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cátions/metabolismo , Simulação de Dinâmica Molecular , Nucleosídeo-Trifosfatase/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Biocatálise , Evolução Biológica , Cátions/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Cinética , Magnésio/química , Magnésio/metabolismo , Nucleosídeo-Trifosfatase/química , Ligação Proteica , Conformação Proteica , Água/química , Água/metabolismo
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