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1.
Signal Transduct Target Ther ; 5(1): 212, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963228

RESUMO

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Síndrome Respiratória Aguda Grave/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/imunologia , Vírion/ultraestrutura
2.
Molecules ; 25(17)2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872217

RESUMO

A pandemic caused by the novel coronavirus (SARS-CoV-2 or COVID-19) began in December 2019 in Wuhan, China, and the number of newly reported cases continues to increase. More than 19.7 million cases have been reported globally and about 728,000 have died as of this writing (10 August 2020). Recently, it has been confirmed that the SARS-CoV-2 main protease (Mpro) enzyme is responsible not only for viral reproduction but also impedes host immune responses. The Mpro provides a highly favorable pharmacological target for the discovery and design of inhibitors. Currently, no specific therapies are available, and investigations into the treatment of COVID-19 are lacking. Therefore, herein, we analyzed the bioactive phytocompounds isolated by gas chromatography-mass spectroscopy (GC-MS) from Tinospora crispa as potential COVID-19 Mpro inhibitors, using molecular docking study. Our analyses unveiled that the top nine hits might serve as potential anti-SARS-CoV-2 lead molecules, with three of them exerting biological activity and warranting further optimization and drug development to combat COVID-19.


Assuntos
Antivirais/química , Betacoronavirus/química , Compostos Fitoquímicos/química , Inibidores de Proteases/química , Tinospora/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/classificação , Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Pandemias , Compostos Fitoquímicos/classificação , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/classificação , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Especificidade por Substrato , Termodinâmica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
3.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
4.
J Transl Med ; 18(1): 321, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831104

RESUMO

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Assuntos
Betacoronavirus/fisiologia , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/genética , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Betacoronavirus/metabolismo , Sítios de Ligação/genética , Infecções por Coronavirus/etnologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Predisposição Genética para Doença/etnologia , Variação Genética , Geografia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etnologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
5.
Science ; 369(6503)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732395

RESUMO

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the ß2 adrenergic receptor (ß2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the ß2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Receptores Adrenérgicos beta 2/química , Receptores de Glucagon/química , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Estrutura Secundária de Proteína
6.
Science ; 369(6503): 524-530, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732418

RESUMO

RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates.


Assuntos
Nucleotidases/química , Ribonucleases/química , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Nucleotidases/genética , Estrutura Secundária de Proteína , Precursores de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Especificidade por Substrato , Proteína 1 de Ligação a X-Box/genética
7.
Life Sci ; 258: 118228, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781071

RESUMO

AIMS: Cyclin-dependent kinase 9 (CDK9) is a member of the CDK subfamily and plays a major role in the regulation of transcriptional elongation. It has attracted widespread attention as a therapeutic target for cancer. Here, we aimed to explore novel CDK 9 inhibitors by using a hybrid virtual screening strategy. MAIN METHODS: A hybrid virtual screening strategy was constructed with computer-aided drug design (CADD). First, compounds were filtered in accordance with Lipinski's rule of five and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Second, a 3D-QSAR pharmacophore model was built and used as a 3D query to screen the obtained hit compounds. Third, the hit compounds were subjected to molecular docking studies. Fourth, molecular dynamics (MD) simulations were performed on CDK9 in complex with the final hits to examine the structural stability. Finally, CDK9 kinase biochemical assay was performed to identify the biological activity of the hit compounds. KEY FINDINGS: Seven hit compounds were screened out. These hit compounds showed drug-like properties in accordance with Lipinski's rule of five and ADMET. Complexes involving the six hit compounds bound to CDK9 exhibited good structural stability in the MD simulation. Furthermore, these six hit compounds had strong inhibitory activity against CDK9 kinase. In particular, hit 3 showed the most promising activity with the percentage of 71%. SIGNIFICANCE: The six hit compounds may be promising novel CDK9 inhibitors, and the hybrid virtual screening strategy designed in this study provides an important reference for the design and synthesis of novel CDK9 inhibitors.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Quinase 9 Dependente de Ciclina/química , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína
8.
J Mol Graph Model ; 100: 107697, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32739642

RESUMO

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound zinc metallopeptidase that generates the vasodilatory peptide angiotensin 1-7 and thus performs a protective role in heart disease. It is considered an important therapeutic target in controlling the COVID-19 outbreak, since SARS-CoV-2 enters permissive cells via an ACE2-mediated mechanism. The present in silico study attempted to repurpose existing drugs for use as prospective viral-entry inhibitors targeting human ACE2. Initially, a clinically approved drug library of 7,173 ligands was screened against the receptor using molecular docking, followed by energy minimization and rescoring of docked ligands. Finally, potential binders were inspected to ensure molecules with different scaffolds were engaged in favorable contacts with both the metal cofactor and the critical residues lining the receptor's active site. The results of the calculations suggest that lividomycin, burixafor, quisinostat, fluprofylline, pemetrexed, spirofylline, edotecarin, and diniprofylline emerge as promising repositionable drug candidates for stabilizing the closed (substrate/inhibitor-bound) conformation of ACE2, thereby shifting the relative positions of the receptor's critical exterior residues recognized by SARS-CoV-2. This study is among the rare ones in the relevant scientific literature to search for potential ACE2 inhibitors. In practical terms, the drugs, unmodified as they are, may be introduced into the therapeutic armamentarium of the ongoing fight against COVID-19 now, or their scaffolds may serve as rich skeletons for designing novel ACE2 inhibitors in the near future.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Antivirais/química , Betacoronavirus/química , Peptidil Dipeptidase A/química , Bibliotecas de Moléculas Pequenas/química , Motivos de Aminoácidos , Betacoronavirus/enzimologia , Carbazóis/química , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Difilina/análogos & derivados , Difilina/química , Interações Hospedeiro-Patógeno , Humanos , Ácidos Hidroxâmicos/química , Ligantes , Simulação de Acoplamento Molecular , Pandemias , Paromomicina/análogos & derivados , Paromomicina/química , Pemetrexede/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Termodinâmica
9.
J Mol Graph Model ; 100: 107690, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32745925

RESUMO

Coronavirus epidemic 2019 (COVID-19), caused by novel coronavirus (2019-nCoV), is newly increasing worldwide and elevating global health concerns. Similar to SARS-CoV and MERS-CoV, the viral key 3-chymotrypsin-like cysteine protease enzyme (3CLPro), which controls 2019-nCoV duplications and manages its life cycle, could be pointed as a drug discovery target. Herein, we theoretically studied the binding ability of 10 structurally different anthocyanins with the catalytic dyad residues of 3CLpro of 2019-nCoV using molecular docking modelling. The results revealed that the polyacylated anthocyanins, including phacelianin, gentiodelphin, cyanodelphin, and tecophilin, were found to authentically bind with the receptor binding site and catalytic dyad (Cys145 and His41) of 2019-nCoV-3CLpro. Our analyses revealed that the top four hits might serve as potential anti-2019-nCoV leading molecules for further optimization and drug development process to combat COVID-19. This study unleashed that anthocyanins with specific structure could be used as effective anti-COVID-19 natural components.


Assuntos
Antocianinas/química , Antivirais/química , Benzopiranos/química , Betacoronavirus/química , Cisteína Endopeptidases/química , Glucosídeos/química , Inibidores de Proteases/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Sítios de Ligação , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores
10.
J Mol Graph Model ; 100: 107710, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32829149

RESUMO

The emergence of SARS-CoV-2 has prompted a worldwide health emergency. There is an urgent need for therapeutics, both through the repurposing of approved drugs and the development of new treatments. In addition to the viral drug targets, a number of human drug targets have been suggested. In theory, targeting human proteins should provide an advantage over targeting viral proteins in terms of drug resistance, which is commonly a problem in treating RNA viruses. This paper focuses on the human protein TMPRSS2, which supports coronavirus life cycles by cleaving viral spike proteins. The three-dimensional structure of TMPRSS2 is not known and so we have generated models of the TMPRSS2 in the apo state as well as in complex with a peptide substrate and putative inhibitors to aid future work. Importantly, many related human proteases have 80% or higher identity with TMPRSS2 in the S1-S1' subsites, with plasminogen and urokinase-type plasminogen activator (uPA) having 95% identity. We highlight 376 approved, investigational or experimental drugs targeting S1A serine proteases that may also inhibit TMPRSS2. Whilst the presence of a relatively uncommon lysine residue in the S2/S3 subsites means that some serine protease inhibitors will not inhibit TMPRSS2, this residue is likely to provide a handle for selective targeting in a focused drug discovery project. We discuss how experimental drugs targeting related serine proteases might be repurposed as TMPRSS2 inhibitors to treat coronaviruses.


Assuntos
Antivirais/química , Betacoronavirus/química , Inibidores de Proteases/química , Serina Endopeptidases/química , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Interações Hospedeiro-Patógeno , Humanos , Ligantes , Simulação de Dinâmica Molecular , Pandemias , Plasminogênio/antagonistas & inibidores , Plasminogênio/química , Plasminogênio/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Serina Endopeptidases/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
11.
Biomol NMR Assign ; 14(2): 339-346, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32803496

RESUMO

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Apoproteínas/química , Betacoronavirus/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Isótopos de Nitrogênio/química , Espectroscopia de Prótons por Ressonância Magnética , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Apoproteínas/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas não Estruturais Virais/metabolismo
12.
Nat Commun ; 11(1): 4137, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811827

RESUMO

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas-G/química , Receptores dos Hormônios Gastrointestinais/química , Secretina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Microscopia Crioeletrônica , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Insetos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/ultraestrutura , Secretina/metabolismo
13.
Science ; 369(6507): 1132-1136, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855341

RESUMO

Naturally occurring proteins vary the precise geometries of structural elements to create distinct shapes optimal for function. We present a computational design method, loop-helix-loop unit combinatorial sampling (LUCS), that mimics nature's ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization showed that 17 (38%) of 45 tested LUCS designs encompassing two different structural topologies were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely matched the designs. LUCS greatly expands the designable structure space and offers a new paradigm for designing proteins with tunable geometries that may be customizable for novel functions.


Assuntos
Desenho Assistido por Computador , Engenharia de Proteínas/métodos , Dobramento de Proteína , Estrutura Secundária de Proteína
14.
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
15.
Int J Antimicrob Agents ; 56(3): 106119, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32738306

RESUMO

Coronavirus disease 2019 (COVID-19) is a highly transmissible viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical trials have reported improved outcomes resulting from an effective reduction or absence of viral load when patients were treated with chloroquine (CQ) or hydroxychloroquine (HCQ). In addition, the effects of these drugs were improved by simultaneous administration of azithromycin (AZM). The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein binds to the cell surface angiotensin-converting enzyme 2 (ACE2) receptor, allowing virus entry and replication in host cells. The viral main protease (Mpro) and host cathepsin L (CTSL) are among the proteolytic systems involved in SARS-CoV-2 S protein activation. Hence, molecular docking studies were performed to test the binding performance of these three drugs against four targets. The findings showed AZM affinity scores (ΔG) with strong interactions with ACE2, CTSL, Mpro and RBD. CQ affinity scores showed three low-energy results (less negative) with ACE2, CTSL and RBD, and a firm bond score with Mpro. For HCQ, two results (ACE2 and Mpro) were firmly bound to the receptors, however CTSL and RBD showed low interaction energies. The differences in better interactions and affinity between HCQ and CQ with ACE2 and Mpro were probably due to structural differences between the drugs. On other hand, AZM not only showed more negative (better) values in affinity, but also in the number of interactions in all targets. Nevertheless, further studies are needed to investigate the antiviral properties of these drugs against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Azitromicina/química , Betacoronavirus/química , Catepsina L/química , Cloroquina/química , Cisteína Endopeptidases/química , Hidroxicloroquina/química , Peptidil Dipeptidase A/química , Glicoproteína da Espícula de Coronavírus/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Antivirais/química , Azitromicina/farmacologia , Betacoronavirus/metabolismo , Sítios de Ligação , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Hidroxicloroquina/farmacologia , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Ligação Viral/efeitos dos fármacos
16.
Nat Commun ; 11(1): 3969, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769976

RESUMO

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg2+-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.


Assuntos
Carboxiliases/metabolismo , Enterococcus faecalis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Carboxiliases/química , Sequência Conservada , Cristalografia por Raios X , Ligantes , Metais/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Especificidade por Substrato
17.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769995

RESUMO

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Tripsina/metabolismo
18.
Front Immunol ; 11: 1663, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754160

RESUMO

A recent pandemic caused by a single-stranded RNA virus, COVID-19, initially discovered in China, is now spreading globally. This poses a serious threat that needs to be addressed immediately. Genome analysis of SARS-CoV-2 has revealed its close relation to SARS-coronavirus along with few changes in its spike protein. The spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. In the current study, the spike protein of SARS-CoV-2 was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. The S1 and S2 domains of spike proteins were analyzed, and two vaccine constructs were prioritized with T-cell and B-cell epitopes. We adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against COVID-19. Prioritized epitopes were then modeled using linkers and adjuvants, and respective 3D models were constructed to evaluate their physiochemical properties and their possible interactions with ACE2, HLA Superfamily alleles, TLR2, and TLR4.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Modelos Químicos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/virologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/imunologia , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/imunologia , Vacinas Virais/química
19.
Food Chem ; 333: 127530, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32683264

RESUMO

Soybean protein isolate (SPI) was incubated with flaxseed gum (FG) at 60 °C for 3 days under high hydrostatic pressure (HHP 0.1-300 MPa). Results showed improvement in solubility of SPI upon glycation with FG. The maximum solubility reached 86.84% when SPI-FG was treated at pH 8.0 and 200 MPa. The occurrence, degrees and sites of SPI-FG glycation suggested that moderate pressure (100 MPa) significantly promoted Maillard reactions, but higher pressures (greater than 200 MPa) suppressed these reactions. The secondary structure of the glycated proteins varied greatly with respect to α-helix and random coil contents and vibrations of the amide II band at 200 MPa. These microstructural changes increased the solubility over a broad pH range. The conformational changes in the glycated SPI supported the improved solubility of SPI-FG. Overall, HHP represents a potential method of controlling glycation to improve protein processability and expand their applicability in the food industry.


Assuntos
Linho/química , Gomas Vegetais/química , Proteínas de Soja/química , Glicosilação , Pressão Hidrostática , Reação de Maillard , Estrutura Secundária de Proteína , Solubilidade
20.
Nat Commun ; 11(1): 3690, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704140

RESUMO

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Eucariotos/metabolismo , Ativação do Canal Iônico , Mecanotransdução Celular , Proteínas de Arabidopsis/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
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