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1.
Nat Commun ; 11(1): 5535, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139697

RESUMO

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.


Assuntos
DNA Helicases/genética , Reparo do DNA , Neoplasias/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Sequência Conservada/genética , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo
2.
Nat Commun ; 11(1): 5405, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106493

RESUMO

Epstein-Barr virus (EBV) is a γ-herpesvirus associated with the occurrence of several human malignancies. BBRF2 and BSRF1 are two EBV tegument proteins that have been suggested to form a hetero-complex and mediate viral envelopment, but the molecular basis of their interaction and the functional mechanism of this complex remains unknown. Here, we present crystal structures of BBRF2 alone and in complex with BSRF1. BBRF2 has a compact globular architecture featuring a central ß-sheet that is surrounded by 10 helices, it represents a novel fold distinct from other known protein structures. The central portion of BSRF1 folds into two tightly associated antiparallel α-helices, forming a composite four-helix bundle with two α-helices from BBRF2 via a massive hydrophobic network. In vitro, a BSRF1-derived peptide binds to BBRF2 and reduces the number of viral genome copies in EBV-positive cells. Exogenous BBRF2 and BSRF1 co-localize at the Golgi apparatus. Furthermore, BBRF2 binds capsid and capsid-associated proteins, whereas BSRF1 associates with glycoproteins. These findings indicate that the BBRF2-BSRF1 complex tethers EBV nucleocapsids to the glycoprotein-enriched Golgi membrane, facilitating secondary envelopment.


Assuntos
Herpesvirus Humano 4/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice
3.
Molecules ; 25(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036293

RESUMO

A novel series of some hydrazones bearing thiazole moiety were generated via solvent-drop grinding of thiazole carbohydrazide 2 with various carbonyl compounds. Also, dehydrative-cyclocondensation of 2 with active methylene compounds or anhydrides gave the respective pyarzole or pyrazine derivatives. The structures of the newly synthesized compounds were established based on spectroscopic evidences and their alternative syntheses. Additionally, the anti-viral activity of all the products was tested against SARS-CoV-2 main protease (Mpro) using molecular docking combined with molecular dynamics simulation (MDS). The average binding affinities of the compounds 3a, 3b, and 3c (-8.1 ± 0.33 kcal/mol, -8.0 ± 0.35 kcal/mol, and -8.2 ± 0.21 kcal/mol, respectively) are better than that of the positive control Nelfinavir (-6.9 ± 0.51 kcal/mol). This shows the possibility of these three compounds to effectively bind to SARS-CoV-2 Mpro and hence, contradict the virus lifecycle.


Assuntos
Antivirais/síntese química , Betacoronavirus/enzimologia , Hidrazonas/síntese química , Inibidores de Proteases/síntese química , Pirazinas/síntese química , Pirazóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/farmacologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Humanos , Hidrazonas/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirazinas/farmacologia , Pirazóis/farmacologia , Termodinâmica , Interface Usuário-Computador , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
4.
Nat Commun ; 11(1): 5175, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056978

RESUMO

DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Replicação do DNA , DNA Fúngico/genética , Evolução Molecular , Mutação , Complexo de Reconhecimento de Origem/ultraestrutura , Conformação Proteica em alfa-Hélice/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/genética
5.
Nat Commun ; 11(1): 5168, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33057011

RESUMO

The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K+ channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fundamental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations.


Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/genética , Canais de Potássio/metabolismo , Potássio/metabolismo , Alanina/genética , Ataxia/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Epilepsia/genética , Humanos , Síndrome do QT Longo/genética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Canais de Potássio/genética , Canais de Potássio/isolamento & purificação , Conformação Proteica em alfa-Hélice/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Streptomyces lividans , Valina/genética
6.
Nat Commun ; 11(1): 5205, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060564

RESUMO

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/química , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Nanismo Hipofisário/genética , Proteínas de Ligação ao GTP , Hormônio Liberador de Hormônio do Crescimento/deficiência , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Transdução de Sinais
7.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024075
8.
PLoS One ; 15(9): e0239836, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986761

RESUMO

Plants are constantly under the threat of various biotic and abiotic stress conditions and to overcome these stresses, they have evolved multiple mechanisms including systematic accumulation of different phytohormones, phytoalexins and pathogenesis related (PR) proteins. PR proteins are cluster of proteins with low molecular weight which get incited in plants under different stresses. In this paper, in-silico approaches are used to compare the physico-chemical properties of 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) of Arabidopsis thaliana and Oryza sativa. Topological analysis revealed the presence of transmembrane localization of PR2 and absence of transmembrane domain in PR10 of both model plants studied. Amino acid composition shows the dominance of small aliphatic amino acids i.e. alanine, glycine and serine in both plants studied. These results highlights the similarities and differences between PRs of both model plants, which provides clue towards their diversified roles in plants.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Simulação por Computador , Oryza/química , Sequência de Aminoácidos , Aminoácidos , Ponto Isoelétrico , Proteínas de Membrana/química , Peso Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos
9.
Molecules ; 25(18)2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927621

RESUMO

Mass spectrometry and some other biophysical methods, have made substantial contributions to the studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins interactions. The most interesting feature of SARS-CoV-2 seems to be the structure of its spike (S) protein and its interaction with the human cell receptor. Mass spectrometry of spike S protein revealed how the glycoforms are distributed across the S protein surface. X-ray crystallography and cryo-electron microscopy made huge impact on the studies on the S protein and ACE2 receptor protein interaction, by elucidating the three-dimensional structures of these proteins and their conformational changes. The findings of the most recent studies in the scope of SARS-CoV-2-Human protein-protein interactions are described here.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/epidemiologia , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/epidemiologia , Receptores Virais/química , Síndrome Respiratória Aguda Grave/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/patogenicidade , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Nucleic Acids Res ; 48(18): 10313-10328, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976585

RESUMO

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a RNA/química , Transcrição Genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Inativação Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Vírus/genética
11.
Molecules ; 25(17)2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32867349

RESUMO

Three types of new coronaviruses (CoVs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and corona-virus disease 2019 (COVID-19). Neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. Each of the 3C-like (3CL) proteases of the three CoVs is essential for the proliferation of the CoVs, and an inhibitor of the 3CL protease (3CLpro) is thought to be an ideal therapeutic agent against SARS, MERS, or COVID-19. Among these, SARS-CoV is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. This article briefly reviews a series of studies on SARS-CoV, focusing on the development of inhibitors for the SARS-CoV 3CLpro based on molecular interactions with the 3CL protease. Our recent approach, based on the structure-based rational design of a novel scaffold for SARS-CoV 3CLpro inhibitor, is also included. The achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including SARS-CoV-2.


Assuntos
Antivirais/química , Betacoronavirus/química , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Inibidores de Proteases/química , Vírus da SARS/patogenicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/uso terapêutico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/genética , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
12.
Nat Commun ; 11(1): 4476, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900995

RESUMO

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.


Assuntos
Engenharia de Proteínas , Multimerização Proteica , Estabilidade Proteica , Actinina/química , Fenômenos Biomecânicos , Conectina/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Desdobramento de Proteína , Imagem Individual de Molécula , Temperatura
13.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
14.
Nucleic Acids Res ; 48(17): 9943-9958, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32821917

RESUMO

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3' side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and ß-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Endonucleases/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Sítios de Ligação , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Fatores de Transcrição/metabolismo
15.
Nucleic Acids Res ; 48(17): 9918-9930, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32785618

RESUMO

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6-4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Oxigenases de Função Mista/química , Pseudomonas putida/enzimologia , RNA de Transferência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , RNA de Transferência/química
16.
Nat Commun ; 11(1): 4033, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820167

RESUMO

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.


Assuntos
Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Idoso , Animais , Linhagem Celular , Desenho de Fármacos , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Ratos , Suínos
17.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
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
19.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776918

RESUMO

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Uridina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Uridina/metabolismo
20.
Molecules ; 25(17)2020 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-32842509

RESUMO

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the Informational spectrum method applied for small molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Mezlocilina/química , Inibidores de Proteases/química , Raltegravir Potássico/química , Proteínas não Estruturais Virais/química , Sítio Alostérico , Antivirais/farmacologia , Betacoronavirus/enzimologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Mezlocilina/farmacologia , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Raltegravir Potássico/farmacologia , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
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