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2.
Biochemistry (Mosc) ; 86(8): 913-925, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488569

RESUMO

Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.


Assuntos
Guanosina Trifosfato/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Bioquímica/história , Catálise , DNA Bacteriano/análise , RNA Polimerases Dirigidas por DNA/química , História do Século XX , Hidrólise , Modelos Moleculares , Dobramento de Proteína , RNA/biossíntese , Ribossomos/fisiologia , U.R.S.S.
3.
Nat Commun ; 12(1): 5393, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518553

RESUMO

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.


Assuntos
Microscopia Crioeletrônica/métodos , Dinamina I/química , Dinamina I/ultraestrutura , Simulação de Dinâmica Molecular , Domínios de Homologia à Plecstrina , Conformação Proteica , Multimerização Proteica , Algoritmos , Dinamina I/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Mutação , Termodinâmica
4.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
5.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
6.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445383

RESUMO

As crucial signal transducers, G-proteins and G-protein-coupled receptors (GPCRs) have attracted increasing attention in the field of signal transduction. Research on G-proteins and GPCRs has mainly focused on animals, while research on plants is relatively rare. The mode of action of G-proteins is quite different from that in animals. The G-protein α (Gα) subunit is the most essential member of the G-protein signal cycle in animals and plants. The G-protein is activated when Gα releases GDP and binds to GTP, and the relationships with the GPCR and the downstream signal are also achieved by Gα coupling. It is important to study the role of Gα in the signaling pathway to explore the regulatory mechanism of G-proteins. The existence of a self-activated Gα in plants makes it unnecessary for the canonical GPCR to activate the G-protein by exchanging GDP with GTP. However, putative GPCRs have been found and proven to play important roles in G-protein signal transduction. The unique mode of action of G-proteins and the function of putative GPCRs in plants suggest that the same definition used in animal research cannot be used to study uncanonical GPCRs in plants. This review focuses on the different functions of the Gα and the mode of action between plants and animals as well as the functions of the uncanonical GPCR. This review employs a new perspective to define uncanonical GPCRs in plants and emphasizes the role of uncanonical GPCRs and Gα subunits in plant stress resistance and agricultural production.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Plantas/metabolismo , Estresse Fisiológico , Animais , Regulação da Expressão Gênica de Plantas , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , Transdução de Sinais
7.
J Phys Chem B ; 125(31): 8805-8813, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34324329

RESUMO

The Ras protein is one of the most important drug targets for battling cancers. To effectively design novel drugs of Ras, we characterize here its conformational ensembles for the hydrolysis intermediate state RasGDP·Pi and the product state RasGDP by extensive replica-exchange molecular dynamics simulations. Several substates for RasGDP·Pi have been identified, while structural analyses have revealed an unrecognized hydrogen-bonding network that stabilizes the hydrolysis intermediate state. More interestingly, Gln61, which is involved in numerous oncogenic mutations, was found to be engaged in this hydrogen-bonding network, adopting a specific conformation that always points to Pi in contrast to that in the RasGTP state. The simulations also reveal that RasGDP has more than one substate, suggesting a conformational selection mechanism for the interaction between Ras and the guanine nucleotide exchange factors (GEFs). These findings offer new opportunities for the drug design of Ras by stabilizing the hydrolysis intermediate or disrupting its interaction with the GEFs.


Assuntos
Hidrogênio , Guanosina Trifosfato , Ligação de Hidrogênio , Hidrólise , Conformação Molecular
8.
J Phys Chem B ; 125(31): 8768-8780, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34328335

RESUMO

The microtubule is regarded as the key target for designing anticancer and neurotherapeutic drugs due to its functional importance in eukaryotic cells including neurons. The microtubule is a dynamic hollow polymer tube consisting of α,ß-tubulin heterodimer. Polymerization of α,ß-tubulin heterodimer resulted in microtubule formation. GTP plays a crucial role in microtubule polymerization. It binds at the exchangeable binding site of the ß-tubulin heterodimer, and it is one of the most crucial therapeutic hot spots for designing anticancer therapeutics. In this manuscript, we have shown using an in silico strategy and various in vitro and cellular experiments that the binding affinity to the tubulin and cancer therapeutic potential of an exchangeable GTP/GDP binding antimitotic tetrapeptide (SP: Ser-Leu-Arg-Pro) is increased through changing proline with the multifluorine substituted proline. This study showcases the importance of the proline amino acid and its pyrrolidine ring in the regulation of binding with tubulin at the GTP binding pocket.


Assuntos
Antimitóticos , Tubulina (Proteína) , Antimitóticos/farmacologia , Sítios de Ligação , Flúor , Guanosina Trifosfato , Microtúbulos/metabolismo , Prolina , Tubulina (Proteína)/metabolismo
9.
Nat Cell Biol ; 23(8): 859-869, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34253896

RESUMO

Dynamin has an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Here, using gold nanorods as cargos to image dynamin action during live clathrin-mediated endocytosis, we show that, near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle increases substantially, accompanied by simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by the coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.


Assuntos
Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Dinaminas/fisiologia , Endocitose/fisiologia , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Microscopia/métodos , Nanotubos , Torque
10.
Braz J Biol ; 82: e250700, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34259718

RESUMO

The mutations are genetic changes in the genome sequences and have a significant role in biotechnology, genetics, and molecular biology even to find out the genome sequences of a cell DNA along with the viral RNA sequencing. The mutations are the alterations in DNA that may be natural or spontaneous and induced due to biochemical reactions or radiations which damage cell DNA. There is another cause of mutations which is known as transposons or jumping genes which can change their position in the genome during meiosis or DNA replication. The transposable elements can induce by self in the genome due to cellular and molecular mechanisms including hypermutation which caused the localization of transposable elements to move within the genome. The use of induced mutations for studying the mutagenesis in crop plants is very common as well as a promising method for screening crop plants with new and enhanced traits for the improvement of yield and production. The utilization of insertional mutations through transposons or jumping genes usually generates stable mutant alleles which are mostly tagged for the presence or absence of jumping genes or transposable elements. The transposable elements may be used for the identification of mutated genes in crop plants and even for the stable insertion of transposable elements in mutated crop plants. The guanine nucleotide-binding (GTP) proteins have an important role in inducing tolerance in rice plants to combat abiotic stress conditions.


Assuntos
Oryza , Elementos de DNA Transponíveis/genética , Expressão Gênica , Guanosina Trifosfato , Oryza/genética , Fenótipo
11.
Molecules ; 26(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208932

RESUMO

We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme-substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate.


Assuntos
Fatores de Ribosilação do ADP/química , Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Proteínas de Membrana/química , Modelos Químicos , Simulação de Dinâmica Molecular , Catálise , Hidrólise
12.
Comput Biol Med ; 135: 104639, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34247129

RESUMO

Mutations in K-Ras are involved in a large number of all human cancers, thus, K-Ras is regarded as a promising target for anticancer drug design. Understanding the target roles of K-Ras is important for providing insights on the molecular mechanism underlying the conformational transformation of the switch domains in K-Ras due to mutations. In this study, multiple replica Gaussian accelerated molecular (MR-GaMD) simulations and principal component analysis (PCA) were applied to probe the effect of G13A, G13D and G13I mutations on conformational transformations of the switch domains in GDP-associated K-Ras. The results suggest that G13A, G13D and G13I enhance the structural flexibility of the switch domains, change the correlated motion modes of the switch domains and strengthen the total motion strength of K-Ras compared with the wild-type (WT) K-Ras. Free energy landscape analyses not only show that the switch domains of the GDP-bound inactive K-Ras mainly exist as a closed state but also indicate that mutations evidently alter the free energy profile of K-Ras and affect the conformational transformation of the switch domains between the closed and open states. Analyses of hydrophobic interaction contacts and hydrogen bonding interactions show that the mutations scarcely change the interaction network of GDP with K-Ras and only disturb the interaction of GDP with the switch (SW1). In summary, two newly introduced mutations, G13A and G13I, play similar adjustment roles in the conformational transformations of two switch domains to G13D and are possibly utilized to tune the activity of K-Ras and the binding of guanine nucleotide exchange factors.


Assuntos
Simulação de Dinâmica Molecular , Proteínas ras , Guanosina Difosfato , Guanosina Trifosfato , Humanos , Análise de Componente Principal , Conformação Proteica , Proteínas ras/metabolismo
13.
J Biol Chem ; 297(1): 100861, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34116056

RESUMO

Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide-loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide-binding domain of RagA or Lys84 of RagC and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.


Assuntos
Aminoácidos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/ultraestrutura , Guanosina Trifosfato/química , Humanos , Ligação de Hidrogênio , Hidrólise , Alvo Mecanístico do Complexo 1 de Rapamicina/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/ultraestrutura , Conformação Proteica , Domínios Proteicos/genética , Multimerização Proteica/genética , Transdução de Sinais/genética
14.
Nat Commun ; 12(1): 3759, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145298

RESUMO

Pol µ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol µ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol µ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol µ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol µ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.


Assuntos
Pareamento Incorreto de Bases/genética , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica/genética , Pareamento de Bases/genética , DNA/química , DNA Ligases/metabolismo , DNA Polimerase Dirigida por DNA/genética , Guanosina Trifosfato/química , Humanos
15.
Atherosclerosis ; 328: 83-91, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118596

RESUMO

BACKGROUND AND AIMS: The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. METHODS: Low-density lipoprotein (LDL) receptor null (LDLr-/-) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. RESULTS: Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. CONCLUSIONS: Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.


Assuntos
Aterosclerose , Óxido Nítrico Sintase Tipo III , Animais , Apolipoproteína A-I , Aterosclerose/tratamento farmacológico , Aterosclerose/prevenção & controle , Biopterina/análogos & derivados , Células Endoteliais , Endotélio Vascular , GTP Cicloidrolase , Guanosina Trifosfato , Camundongos , Óxido Nítrico , Peptídeos , Superóxidos
16.
FEBS Lett ; 595(14): 1876-1885, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060653

RESUMO

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestrutura
17.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
18.
J Mol Biol ; 433(15): 167046, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-33971210

RESUMO

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.


Assuntos
Archaea/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Mutação , Fatores de Alongamento de Peptídeos/genética , Conformação Proteica , Domínios Proteicos
19.
Dev Biol ; 478: 89-101, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34048735

RESUMO

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.


Assuntos
Estruturas Citoplasmáticas/ultraestrutura , IMP Desidrogenase/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Estruturas Citoplasmáticas/química , Expressão Gênica , Guanosina Trifosfato/biossíntese , Guanosina Trifosfato/metabolismo , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Isoenzimas/química , Isoenzimas/genética , Mutação Puntual , Regulação para Cima , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
J Mol Biol ; 433(15): 167061, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34023403

RESUMO

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.


Assuntos
4-Hidroxicumarinas/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Mycobacterium tuberculosis/metabolismo , 4-Hidroxicumarinas/química , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice
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