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1.
Nat Commun ; 11(1): 5538, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139716

RESUMO

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.


Assuntos
Fosfotransferases (Fosfomutases)/metabolismo , Processamento de Proteína Pós-Traducional , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Ensaios Enzimáticos , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glicólise , Isomerismo , Cinética , Conformação Molecular , Fosforilação , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/isolamento & purificação , Fosfotransferases (Fosfomutases)/ultraestrutura , Prolina/química , Domínios Proteicos , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
Nat Commun ; 11(1): 4948, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009415

RESUMO

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.


Assuntos
Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperonas Moleculares/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulação Alostérica , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Transporte Biológico , Modelos Moleculares , Domínios Proteicos
3.
Sci Rep ; 10(1): 17699, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077836

RESUMO

Angiotensin converting enzyme 2 (ACE2) (EC:3.4.17.23) is a transmembrane protein which is considered as a receptor for spike protein binding of novel coronavirus (SARS-CoV2). Since no specific medication is available to treat COVID-19, designing of new drug is important and essential. In this regard, in silico method plays an important role, as it is rapid and cost effective compared to the trial and error methods using experimental studies. Natural products are safe and easily available to treat coronavirus affected patients, in the present alarming situation. In this paper five phytochemicals, which belong to flavonoid and anthraquinone subclass, have been selected as small molecules in molecular docking study of spike protein of SARS-CoV2 with its human receptor ACE2 molecule. Their molecular binding sites on spike protein bound structure with its receptor have been analyzed. From this analysis, hesperidin, emodin and chrysin are selected as competent natural products from both Indian and Chinese medicinal plants, to treat COVID-19. Among them, the phytochemical hesperidin can bind with ACE2 protein and bound structure of ACE2 protein and spike protein of SARS-CoV2 noncompetitively. The binding sites of ACE2 protein for spike protein and hesperidin, are located in different parts of ACE2 protein. Ligand spike protein causes conformational change in three-dimensional structure of protein ACE2, which is confirmed by molecular docking and molecular dynamics studies. This compound modulates the binding energy of bound structure of ACE2 and spike protein. This result indicates that due to presence of hesperidin, the bound structure of ACE2 and spike protein fragment becomes unstable. As a result, this natural product can impart antiviral activity in SARS CoV2 infection. The antiviral activity of these five natural compounds are further experimentally validated with QSAR study.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Antraquinonas/química , Antraquinonas/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Emodina/química , Emodina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química
4.
Nucleic Acids Res ; 48(17): 9969-9985, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32974652

RESUMO

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.


Assuntos
Sítio Alostérico , Elementos de Resposta , Receptores X Retinoide/química , Regulação Alostérica , DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores X Retinoide/metabolismo
5.
Mol Pharmacol ; 98(4): 303-313, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32873746

RESUMO

Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria and a potent orthosteric agonist of the GABAA receptor. The binding of [3H]muscimol has been used to evaluate the distribution of GABAA receptors in the brain, and studies of modulation of [3H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABAA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor. Here, we employed the Monod-Wyman-Changeux concerted transition model to analyze muscimol binding isotherms. We show that the binding isotherms from recombinant α1ß3 GABAA receptors can be qualitatively predicted using electrophysiological data pertaining to properties of receptor activation and desensitization in the presence of muscimol. The model predicts enhancement of [3H]muscimol binding in the presence of the steroids allopregnanolone and pregnenolone sulfate, although the steroids interact with distinct sites and either enhance (allopregnanolone) or reduce (pregnenolone sulfate) receptor function. We infer that the concerted transition model can be used to link radioligand binding and electrophysiological data. SIGNIFICANCE STATEMENT: The study employs a three-state resting-active-desensitized model to link radioligand binding and electrophysiological data. We show that the binding isotherms can be qualitatively predicted using parameters estimated in electrophysiological experiments and that the model accurately predicts the enhancement of [3H]muscimol binding in the presence of the potentiating steroid allopregnanolone and the inhibitory steroid pregnenolone sulfate.


Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Muscimol/farmacologia , Receptores de GABA-A/metabolismo , Esteroides/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Células HEK293 , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Muscimol/química , Pregnanolona/farmacologia , Pregnenolona/farmacologia , Receptores de GABA-A/química , Receptores de GABA-A/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trítio/química
6.
Life Sci ; 261: 118455, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32956662

RESUMO

Diabetic nephropathy (DN) is a chronic complication of diabetes mellitus (DM) with approximately 30-40% of patients with DM developing nephropathy, and it is the leading cause of end-stage renal diseases and diabetic morbidity. The pathogenesis of DN is primarily associated with irregularities in the metabolism of glucose and lipid leading to hyperglycemia-induced oxidative stress, which has been a major target together with blood pressure regulation in the control of DN progression. However, the regulation of 5' adenosine monophosphate-activated protein kinase (AMPK), a highly conserved protein kinase for maintaining energy balance and cellular growth and repair has been implicated in the development of DM and its complications. Therefore, targeting AMPK pathway has been explored as a therapeutic strategy for the treatment of diabetes and its complication, although most of the mechanisms have not been fully elucidated. In this review, we discuss the structure of AMPK relevant to understanding its allosteric regulation and its role in the pathogenesis and progression of DN. We also identify therapeutic agents that modulate AMPK and its downstream targets with their specific mechanisms of action in the treatment of DN.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação Alostérica/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Descoberta de Drogas , Transdução de Sinais/efeitos dos fármacos , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Humanos , Terapia de Alvo Molecular
7.
PLoS One ; 15(9): e0222548, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32870913

RESUMO

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-É£) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-É£, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-É£ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.


Assuntos
Linfócitos B/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Transferência Ressonante de Energia de Fluorescência , Voluntários Saudáveis , Humanos , Injeções Intraperitoneais , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Fenotiazinas/farmacologia , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo
8.
Nature ; 585(7824): 303-308, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879488

RESUMO

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.


Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Barbitúricos/química , Barbitúricos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Microscopia Crioeletrônica , Receptores de GABA-A/química , Regulação Alostérica/efeitos dos fármacos , Anestésicos Gerais/metabolismo , Barbitúricos/metabolismo , Benzodiazepinas/metabolismo , Bicuculina/química , Bicuculina/metabolismo , Bicuculina/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Diazepam/química , Diazepam/metabolismo , Diazepam/farmacologia , Eletrofisiologia , Etomidato/química , Etomidato/metabolismo , Etomidato/farmacologia , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Fenobarbital/química , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Picrotoxina/química , Picrotoxina/metabolismo , Picrotoxina/farmacologia , Propofol/química , Propofol/metabolismo , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestrutura , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
9.
Proc Natl Acad Sci U S A ; 117(35): 21711-21722, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817431

RESUMO

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+ alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+ activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.


Assuntos
Cálcio/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/ultraestrutura , Regulação Alostérica/fisiologia , Sítios de Ligação/genética , Cálcio/fisiologia , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Ligantes , Conformação Molecular , Receptores de Detecção de Cálcio/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais
10.
Nat Commun ; 11(1): 3862, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737291

RESUMO

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.


Assuntos
Algoritmos , Proteínas Quimiotáticas Aceptoras de Metil/química , Mapeamento de Interação de Proteínas/estatística & dados numéricos , Software , Regulação Alostérica , Sítio Alostérico , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Internet , Proteínas Quimiotáticas Aceptoras de Metil/antagonistas & inibidores , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
11.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
12.
Nat Commun ; 11(1): 4135, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811817

RESUMO

Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.


Assuntos
Complexo I de Transporte de Elétrons/química , Simulação de Dinâmica Molecular , Quinonas/química , Thermus thermophilus/enzimologia , Regulação Alostérica , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/ultraestrutura , Modelos Moleculares , NAD/química , NAD/metabolismo , Redes Neurais de Computação , Conformação Proteica , Prótons , Quinonas/metabolismo , Thermus thermophilus/genética
13.
Proc Natl Acad Sci U S A ; 117(36): 22080-22089, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820071

RESUMO

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.


Assuntos
Ácidos Graxos/metabolismo , Lisofosfatidilcolinas/metabolismo , Palmitoil-CoA Hidrolase/química , Palmitoil-CoA Hidrolase/metabolismo , Acil Coenzima A/metabolismo , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Regulação Alostérica , Ácidos Graxos/química , Humanos , Cinética , Gotículas Lipídicas/enzimologia , Gotículas Lipídicas/metabolismo , Lisofosfatidilcolinas/química , Palmitoil-CoA Hidrolase/genética , Domínios Proteicos
14.
Proc Natl Acad Sci U S A ; 117(34): 20586-20596, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778600

RESUMO

While recent experiments revealed that some pioneer transcription factors (TFs) can bind to their target DNA sequences inside a nucleosome, the binding dynamics of their target recognitions are poorly understood. Here we used the latest coarse-grained models and molecular dynamics simulations to study the nucleosome-binding procedure of the two pioneer TFs, Sox2 and Oct4. In the simulations for a strongly positioning nucleosome, Sox2 selected its target DNA sequence only when the target was exposed. Otherwise, Sox2 entropically bound to the dyad region nonspecifically. In contrast, Oct4 plastically bound on the nucleosome mainly in two ways. First, the two POU domains of Oct4 separately bound to the two parallel gyres of the nucleosomal DNA, supporting the previous experimental results of the partial motif recognition. Second, the POUS domain of Oct4 favored binding on the acidic patch of histones. Then, simulating the TFs binding to a genomic nucleosome, the LIN28B nucleosome, we found that the recognition of a pseudo motif by Sox2 induced the local DNA bending and shifted the population of the rotational position of the nucleosomal DNA. The redistributed DNA phase, in turn, changed the accessibility of a distant TF binding site, which consequently affected the binding probability of a second Sox2 or Oct4. These results revealed a nucleosomal DNA-mediated allosteric mechanism, through which one TF binding event can change the global conformation, and effectively regulate the binding of another TF at distant sites. Our simulations provide insights into the binding mechanism of single and multiple TFs on the nucleosome.


Assuntos
Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regulação Alostérica , Humanos , Simulação de Dinâmica Molecular , Proteínas de Ligação a RNA/genética
15.
Nucleic Acids Res ; 48(13): 7520-7531, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32678884

RESUMO

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Sequência Consenso , RNA/química , 2',5'-Oligoadenilato Sintetase/química , Células A549 , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , RNA/metabolismo
16.
Mol Pharmacol ; 98(4): 292-302, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690627

RESUMO

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4ß2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.


Assuntos
Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutação , Estereoisomerismo , Sulfonamidas/química , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética
17.
Nucleic Acids Res ; 48(14): 7728-7747, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609811

RESUMO

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.


Assuntos
Processamento Alternativo , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Código das Histonas , Humanos , Camundongos , Ligação Proteica , Domínio Tudor , Ubiquitina-Proteína Ligases/genética
18.
Nucleic Acids Res ; 48(14): 7844-7855, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652013

RESUMO

The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.


Assuntos
Aurora Quinase A/fisiologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Regulação Alostérica , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HeLa , Humanos , Interfase/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Origem de Replicação
19.
Proc Natl Acad Sci U S A ; 117(31): 18459-18469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694211

RESUMO

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
20.
ChemMedChem ; 15(18): 1682-1690, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32663362

RESUMO

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Betacoronavirus/química , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Regulação Alostérica , Inibidores da Enzima Conversora de Angiotensina/química , Domínio Catalítico , Humanos , Peptidil Dipeptidase A/química , Ligação Proteica , Domínios Proteicos , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química
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