Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.410
Filtrar
1.
Nat Commun ; 11(1): 3658, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694613

RESUMO

Biological systems organize multiple hierarchical structures in parallel, and create dynamic assemblies and functions by energy dissipation. In contrast, emerging artificial non-equilibrium self-assembling systems have remained relatively simplistic concerning hierarchical design, and non-equilibrium multi-component systems are uncharted territory. Here we report a modular DNA toolbox allowing to program transient non-equilibrium multicomponent systems across hierarchical length scales by introducing chemically fueled molecular recognition orchestrated by reaction networks of concurrent ATP-powered ligation and cleavage of freely programmable DNA building blocks. Going across hierarchical levels, we demonstrate transient side-chain functionalized nucleic acid polymers, and further introduce the concept of transient cooperative multivalency as a key to bridge length scales to pioneer fuel-driven encapsulation, self-assembly of colloids, and non-equilibrium transient narcissistic colloidal self-sorting on a systems level. The fully programmable and functionalizable DNA components pave the way to design chemically fueled 4D (3 space, 1 time) molecular multicomponent systems and autonomous materials.


Assuntos
Trifosfato de Adenosina/química , Bioengenharia/métodos , DNA/química , Nanotecnologia/métodos , Coloides , DNA Ligases/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Conformação de Ácido Nucleico , Polimerização , Polímeros/química
2.
PLoS Comput Biol ; 16(7): e1007996, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667909

RESUMO

Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Potenciais da Membrana , Mitocôndrias/metabolismo , Vasoconstrição , Trifosfato de Adenosina/química , Cálcio/química , Fosfatos de Cálcio/química , Córtex Cerebral/fisiopatologia , Circulação Cerebrovascular , Citosol/química , Retículo Endoplasmático/química , Substância Cinzenta/fisiopatologia , Humanos , Modelos Teóricos , Acoplamento Neurovascular , Oscilometria , Oxigênio/química , Fosforilação , ATPases Translocadoras de Prótons/química , Acidente Vascular Cerebral/fisiopatologia
3.
PLoS Comput Biol ; 16(7): e1008110, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716928

RESUMO

The concept of minimal cut sets (MCS) provides a flexible framework for analyzing properties of metabolic networks and for computing metabolic intervention strategies. In particular, it has been used to support the targeted design of microbial strains for bio-based production processes. Herein we present a number of major extensions that generalize the existing MCS approach and broaden its scope for applications in metabolic engineering. We first introduce a modified approach to integrate gene-protein-reaction associations (GPR) in the metabolic network structure for the computation of gene-based intervention strategies. In particular, we present a set of novel compression rules for GPR associations, which effectively speedup the computation of gene-based MCS by a factor of up to one order of magnitude. These rules are not specific for MCS and as well applicable to other computational strain design methods. Second, we enhance the MCS framework by allowing the definition of multiple target (undesired) and multiple protected (desired) regions. This enables precise tailoring of the metabolic solution space of the designed strain with unlimited flexibility. Together with further generalizations such as individual cost factors for each intervention, direct combinations of reaction/gene deletions and additions as well as the possibility to search for substrate co-feeding strategies, the scope of the MCS framework could be broadly extended. We demonstrate the applicability and performance benefits of the described developments by computing (gene-based) Escherichia coli strain designs for the bio-based production of 2,3-butanediol, a chemical, that has recently received much attention in the field of metabolic engineering. With our extended framework, we could identify promising strain designs that were formerly unpredictable, including those based on substrate co-feeding.


Assuntos
Escherichia coli/genética , Deleção de Genes , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Trifosfato de Adenosina/química , Aerobiose , Algoritmos , Butileno Glicóis/farmacologia , Simulação por Computador , Microbiologia Industrial , Modelos Biológicos , Modelos Estatísticos , Oxirredução , Processos Estocásticos
4.
Nat Commun ; 11(1): 2911, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518308

RESUMO

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.


Assuntos
Difosfato de Adenosina/química , Culex/química , Mosquitos Vetores , Proteínas e Peptídeos Salivares/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Biologia Computacional/métodos , Cristalografia por Raios X , Eicosanoides/química , Comportamento Alimentar , Perfilação da Expressão Gênica , Hemostasia , Humanos , Proteínas de Insetos/química , Ligantes , Nucleotídeos/química , Agregação Plaquetária , Ligação Proteica , Domínios Proteicos , Saliva/química , Termodinâmica
5.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32521159

RESUMO

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/metabolismo , Betacoronavirus/enzimologia , Inibidores Enzimáticos/metabolismo , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Antivirais/química , Sítios de Ligação , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , RNA Replicase/química , Termodinâmica , Proteínas não Estruturais Virais/química
6.
Nucleic Acids Res ; 48(12): 6640-6653, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449930

RESUMO

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.


Assuntos
DNA Helicases/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Quadruplex G , Recombinases Rec A/química , Trifosfato de Adenosina/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligantes , Conformação de Ácido Nucleico , Recombinases Rec A/genética
7.
Nucleic Acids Res ; 48(12): 6980-6995, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453425

RESUMO

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/ultraestrutura , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Conformação Proteica
8.
Mol Biol (Mosk) ; 54(2): 300-307, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392200

RESUMO

The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated enzymes is carried out both in vitro and in vivo, in the presence of chaperones, usually at temperature optimal for the particular enzyme for the manifestation of enzymatic activity. In the present work thermal stability of enzymes in vitro (using purified preparations) and in vivo (directly in the bacterial cell) has been determined. Bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that the thermal stability of the P. luminescens and P. leiognathi luciferases in vivo in the Escherichia coli MG1655 dnaK^(+) and PK202 ΔdnaKJ14 strains is considerable higher than the thermal stability of "cell-free extract" luciferases. When an uncoupler of oxidative phosphorylation the carbonyl-cyanide-3-chlorophenylhydrazone (CCCP) that reduce the intracellular concentration of ATP to a minimum level, and the volatile hydrophobic substance (-)-Limonene (C10H16) as an inhibitor of chaperone-dependent refolding are added to the medium, the thermal stability of luciferases reduces almost to the level which is characteristic for the purified protein preparation. It is shown that the ATP-dependent chaperones ClpA and ClpB are essential for the increase of thermostability of luciferases in bacterial cells. Also, it is shown that the DnaKJE-dependent refolding of thermoinactivated luciferases is practically absent if the protonophore СССР or the hydrophobic substance (-)-Limonene was added to the bacterial suspension. Taking the data presented in this paper into account, it is necessary to consider the presence in bacterial cells of two different groups of ATP-dependent chaperones: 1st group (DnaKJE, GroEL/ES) is able to conduct the refolding both at low temperature after protein thermal inactivation and at high temperature at which protein thermal inactivation occurs; 2nd group (ClpA,ClpB, and possibly still unknown chaperones) is unable to conduct the standard refolding (i.e. at low temperature), but capable due to the hydrolysis energy of ATP of maintaining nonequilibrium stabilization of protein native forms at high temperature.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Chaperonas Moleculares/química , Dobramento de Proteína , Endopeptidase Clp , Estabilidade Proteica , Temperatura
9.
Nucleic Acids Res ; 48(12): 6471-6480, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442296

RESUMO

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.


Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Simulação de Dinâmica Molecular , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Ligação de Hidrogênio , Mutação
10.
Nat Commun ; 11(1): 2422, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415118

RESUMO

Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Genética , Trifosfato de Adenosina/química , Teorema de Bayes , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrólise , Cinética , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , RNA Mensageiro/metabolismo , Fator sigma/metabolismo , Análise de Célula Única , Processos Estocásticos
11.
Nucleic Acids Res ; 48(10): 5603-5615, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32315072

RESUMO

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.


Assuntos
Alanina , Substituição de Aminoácidos , Lisina/química , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Domínio Catalítico , Lisina/metabolismo , Manganês/química , Modelos Moleculares , Naegleria/enzimologia , RNA Ligase (ATP)/metabolismo
12.
Phys Rev Lett ; 124(11): 118002, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32242707

RESUMO

Motivated by the dynamics of particles embedded in active gels, both in vitro and inside the cytoskeleton of living cells, we study an active generalization of the classical trap model. We demonstrate that activity leads to dramatic modifications in the diffusion compared to the thermal case: the mean square displacement becomes subdiffusive, spreading as a power law in time, when the trap depth distribution is a Gaussian and is slower than any power law when it is drawn from an exponential distribution. The results are derived for a simple, exactly solvable, case of harmonic traps. We then argue that the results are robust for more realistic trap shapes when the activity is strong.


Assuntos
Biopolímeros/química , Modelos Químicos , Citoesqueleto de Actina/química , Trifosfato de Adenosina/química , Difusão , Géis/química , Miosina Tipo II/química
13.
Nat Struct Mol Biol ; 27(4): 373-381, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231289

RESUMO

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/ultraestrutura , Conexinas/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Tecido Nervoso/ultraestrutura , Trifosfato de Adenosina/genética , Animais , Anuros/genética , Membrana Celular/química , Membrana Celular/genética , Conexinas/química , Conexinas/genética , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Transdução de Sinais/genética
14.
Nat Commun ; 11(1): 1916, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317635

RESUMO

mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Mitocôndrias/química , Proteínas Mitocondriais/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Citosol/química , Humanos , Ligação de Hidrogênio , Hidrólise , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína
15.
Proc Natl Acad Sci U S A ; 117(14): 7814-7823, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198203

RESUMO

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an "ADP-like" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Conformação Proteica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Transferência Ressonante de Energia de Fluorescência , Heterogeneidade Genética , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Humanos , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica/genética , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica/genética
16.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
17.
Nat Commun ; 11(1): 1410, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179743

RESUMO

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.


Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Biocatálise , Proteínas de Choque Térmico HSP90/genética , Hidrólise , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Peixe-Zebra/genética
18.
Nat Struct Mol Biol ; 27(4): 323-332, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203490

RESUMO

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
19.
Chemistry ; 26(28): 6279-6284, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32154932

RESUMO

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.


Assuntos
Trifosfato de Adenosina/química , Corantes Fluorescentes/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Enzimas Ativadoras de Ubiquitina/química
20.
Nat Struct Mol Biol ; 27(3): 233-239, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066964

RESUMO

Genome regulation requires control of chromosome organization by SMC-kleisin complexes. The cohesin complex contains the Smc1 and Smc3 subunits that associate with the kleisin Scc1 to form a ring-shaped complex that can topologically engage chromatin to regulate chromatin structure. Release from chromatin involves opening of the ring at the Smc3-Scc1 interface in a reaction that is controlled by acetylation and engagement of the Smc ATPase head domains. To understand the underlying molecular mechanisms, we have determined the 3.2-Šresolution cryo-electron microscopy structure of the ATPγS-bound, heterotrimeric cohesin ATPase head module and the 2.1-Šresolution crystal structure of a nucleotide-free Smc1-Scc1 subcomplex from Saccharomyces cerevisiae and Chaetomium thermophilium. We found that ATP-binding and Smc1-Smc3 heterodimerization promote conformational changes within the ATPase that are transmitted to the Smc coiled-coil domains. Remodeling of the coiled-coil domain of Smc3 abrogates the binding surface for Scc1, thus leading to ring opening at the Smc3-Scc1 interface.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaetomium/química , Chaetomium/genética , Chaetomium/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA