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1.
Biomed Res Int ; 2020: 5324560, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029513

RESUMO

The ongoing global pandemic caused by the human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions of people and claimed hundreds of thousands of lives. The absence of approved therapeutics to combat this disease threatens the health of all persons on earth and could cause catastrophic damage to society. New drugs are therefore urgently required to bring relief to people everywhere. In addition to repurposing existing drugs, natural products provide an interesting alternative due to their widespread use in all cultures of the world. In this study, alkaloids from Cryptolepis sanguinolenta have been investigated for their ability to inhibit two of the main proteins in SARS-CoV-2, the main protease and the RNA-dependent RNA polymerase, using in silico methods. Molecular docking was used to assess binding potential of the alkaloids to the viral proteins whereas molecular dynamics was used to evaluate stability of the binding event. The results of the study indicate that all 13 alkaloids bind strongly to the main protease and RNA-dependent RNA polymerase with binding energies ranging from -6.7 to -10.6 kcal/mol. In particular, cryptomisrine, cryptospirolepine, cryptoquindoline, and biscryptolepine exhibited very strong inhibitory potential towards both proteins. Results from the molecular dynamics study revealed that a stable protein-ligand complex is formed upon binding. Alkaloids from Cryptolepis sanguinolenta therefore represent a promising class of compounds that could serve as lead compounds in the search for a cure for the corona virus disease.


Assuntos
Alcaloides/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Cryptolepis/química , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/antagonistas & inibidores , Alcaloides/química , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/enzimologia , Simulação por Computador , Infecções por Coronavirus/virologia , Cisteína Endopeptidases , Avaliação Pré-Clínica de Medicamentos , Humanos , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/virologia , Relação Quantitativa Estrutura-Atividade , Quinolinas/química , Quinolinas/farmacologia , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores
2.
PLoS One ; 15(10): e0240004, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002032

RESUMO

The SARS-CoV-2 virus has caused a pandemic and is public health emergency of international concern. As of now, no registered therapies are available for treatment of coronavirus infection. The viral infection depends on the attachment of spike (S) glycoprotein to human cell receptor angiotensin-converting enzyme 2 (ACE2). We have designed a protein inhibitor (ΔABP-D25Y) targeting S protein using computational approach. The inhibitor consists of two α helical peptides homologues to protease domain (PD) of ACE2. Docking studies and molecular dynamic simulation revealed that the inhibitor binds exclusively at the ACE2 binding site of S protein. The computed binding affinity of the inhibitor is higher than the ACE2 and thus will likely out compete ACE2 for binding to S protein. Hence, the proposed inhibitor ΔABP-D25Y could be a potential blocker of S protein and receptor binding domain (RBD) attachment.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Desenho de Fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Homologia Estrutural de Proteína , Sítios de Ligação , Simulação por Computador , Infecções por Coronavirus , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral , Domínios Proteicos
3.
Int J Mol Sci ; 21(19)2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33036230

RESUMO

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Virais Reguladoras e Acessórias/química , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homologia de Sequência , Proteínas Virais Reguladoras e Acessórias/metabolismo , Dedos de Zinco
4.
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
5.
Nat Commun ; 11(1): 4916, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004803

RESUMO

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.


Assuntos
Brassica rapa/fisiologia , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Animais , Cristalografia , Flores/metabolismo , Haplótipos , Simulação de Dinâmica Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Pólen/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera
6.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2235-2238, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018452

RESUMO

Electroporation is a well-established technique used to stimulate cells, enhancing membrane permeability. Although the biological phenomena occurring after the poration process have been widely studied, the physical mechanisms of pore formation are not clearly understood. In this work we investigated by means of molecular dynamics simulations the kinetics of pore formation, linking the different stages of poration to specific arrangements of lipid membrane domains.Clinical Relevance-The approach followed in this study aims to shed light on the molecular mechanisms at the basis of the electroporation technique, nowadays used to enhance the entrance of poorly permeant anticancer drugs into tumor cells, for gene electrotransfer and all the other applications exploiting the modulation of cell membrane properties.


Assuntos
Fenômenos Biológicos , Bicamadas Lipídicas , Eletroporação , Cinética , Simulação de Dinâmica Molecular
7.
J Chem Phys ; 153(11): 115101, 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-32962355

RESUMO

Broad-spectrum antiviral drugs are urgently needed to stop the Coronavirus Disease 2019 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines, and their protonation/deprotonation modulates catalysis and conformational plasticity. Here, we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using the recently developed graphical processing unit (GPU)-accelerated implicit-solvent continuous constant pH molecular dynamics method with a new asynchronous replica-exchange scheme, which allows computation on a single GPU card. The calculated pKa's support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 can open and close depending on the protonation state of C271/270, consistent with the most recent crystal structure evidence. Interestingly, despite the lack of an analogous cysteine, BL2 in MERS-CoV PLpro is also very flexible, challenging a current hypothesis. These findings are supported by the all-atom fixed-charge simulations and provide a starting point for more detailed studies to assist the structure-based design of broad-spectrum inhibitors against CoV PLpros.


Assuntos
Antivirais/farmacologia , Betacoronavirus/enzimologia , Desenho de Fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Simulação de Dinâmica Molecular , Papaína/química , Papaína/metabolismo , Prótons , Sequência de Aminoácidos , Histidina , Concentração de Íons de Hidrogênio , Papaína/antagonistas & inibidores , Domínios Proteicos
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.
Chemosphere ; 254: 126918, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32957302

RESUMO

The increasing application of various surfactants nowadays, may lead to the contamination of the natural environment and represent potential threat to terrestrial higher plants. In this article, the effect of 13 surfactants, with dodecyl alkyl chain and various aromatic (imidazolium, pyridinium, thiazolium) and aliphatic (guanidinium, ammonium, thiosemicarbazidium) polar heads, on germination, development and growth of wheat and cucumber was investigated. The study aimed to prove how changes in lipophilicity of surfactants and their various structural modifications (existence of the aliphatic or aromatic polar group, the introduction of oxygen and sulfur) influence toxicity towards investigated plants. The calculated lipophilic parameter (AlogP) is shown to be a useful parameter for predicting potential toxicity of the compound. The strategy of using surfactants with aliphatic polar heads instead of aromatic prove to be a promising strategy in reducing harmful effect, as well as the introduction of polar groups in the structure of cation. From all investigated compounds, surfactants with imidazolium polar head displayed the most harmful effect towards wheat and cucumber. The cucumber seeds were more sensitive to the addition of surfactants comparing to wheat. All obtained experimental results were additionally investigated using computational methods, simulating the transport of surfactants through a lipid bilayer. The influence of cation tendency to fit in lipid bilayer structure was correlated with toxicity. For the first time, it is concluded that cation ability to mimic the structure of bilayer have less harmful effect on plant development.


Assuntos
Cucumis sativus/efeitos dos fármacos , Imidazóis/toxicidade , Compostos de Piridínio/toxicidade , Tensoativos/toxicidade , Triticum/efeitos dos fármacos , Cátions , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Germinação/efeitos dos fármacos , Imidazóis/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tensoativos/química , Triticum/crescimento & desenvolvimento
10.
PLoS Comput Biol ; 16(9): e1007815, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925900

RESUMO

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas dos Microfilamentos , Proteínas de Saccharomyces cerevisiae , Domínios de Homologia de src , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínios de Homologia de src/genética , Domínios de Homologia de src/fisiologia
11.
PLoS Comput Biol ; 16(9): e1007922, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946455

RESUMO

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. Since time-resolved biophysical experiments are currently unable to provide full reconstruction of the physico-chemical mechanisms responsible for prion replication, one must rely on computer simulations. In this work, we show that a recently developed algorithm called Self-Consistent Path Sampling (SCPS) overcomes the computational limitations of plain MD and provides a viable tool to investigate prion replication processes using state-of-the-art all-atom force fields in explicit solvent. First, we validate the reliability of SCPS simulations by characterizing the folding of a class of small proteins and comparing against the results of plain MD simulations. Next, we use SCPS to investigate the replication of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Our atomistic reconstruction shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained using a simpler and more approximate path sampling algorithm. Together, these results suggest that the propagation of prions generated by evolutionary distant proteins may share common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.


Assuntos
Proteínas Fúngicas , Simulação de Dinâmica Molecular , Príons , Algoritmos , Biologia Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Príons/química , Príons/metabolismo , Conformação Proteica , Dobramento de Proteína
12.
Nat Commun ; 11(1): 4808, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968058

RESUMO

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.


Assuntos
Simulação por Computador , Evolução Molecular Direcionada/métodos , Enzimas/química , Evolução Química , Liases/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Enzimas/genética , Enzimas/metabolismo , Cinética , Liases/genética , Liases/metabolismo , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Engenharia de Proteínas
13.
Nat Commun ; 11(1): 4800, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968072

RESUMO

Out-of-equilibrium molecular systems hold great promise as dynamic, reconfigurable matter that executes complex tasks autonomously. However, translating molecular scale dynamics into spatiotemporally controlled phenomena emerging at mesoscopic scale remains a challenge-especially if one aims at a design where the system itself maintains gradients that are required to establish spatial differentiation. Here, we demonstrate how surface tension gradients, facilitated by a linear amphiphile molecule, generate Marangoni flows that coordinate the positioning of amphiphile source and drain droplets floating at air-water interfaces. Importantly, at the same time, this amphiphile leads, via buckling instabilities in lamellar systems of said amphiphile, to the assembly of millimeter long filaments that grow from the source droplets and get absorbed at the drain droplets. Thereby, the Marangoni flows and filament organization together sustain the autonomous positioning of interconnected droplet-filament networks at the mesoscale. Our concepts provide potential for the development of non-equilibrium matter with spatiotemporal programmability.


Assuntos
Citoesqueleto/química , Bainha de Mielina/química , Fenômenos Físicos , Cinética , Microscopia , Simulação de Dinâmica Molecular , Tensão Superficial , Água/química
14.
J Chem Theory Comput ; 16(10): 6383-6396, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-32905698

RESUMO

Molecular dynamics simulations are a popular means to study biomolecules, but it is often difficult to gain insights from the trajectories due to their large size, in both time and number of features. The Sapphire (States And Pathways Projected with HIgh REsolution) plot allows a direct visual inference of the dominant states visited by high-dimensional systems and how they are interconnected in time. Here, we extend this visual inference into a clustering algorithm. Specifically, the automatic procedure derives from the Sapphire plot states that are kinetically homogeneous, structurally annotated, and of tunable granularity. We provide a relative assessment of the kinetic fidelity of the Sapphire-based partitioning in comparison to popular clustering methods. This assessment is carried out on trajectories of n-butane, a ß-sheet peptide, and the small protein BPTI. We conclude with an application of our approach to a recent 100 µs trajectory of the main protease of SARS-CoV-2.


Assuntos
Butanos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Proteínas/química , Algoritmos , Betacoronavirus/química , Análise por Conglomerados , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Humanos , Cinética , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Proteínas não Estruturais Virais/química
15.
J Immunol Res ; 2020: 2837670, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32964056

RESUMO

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has previously never been identified with humans, thereby creating devastation in public health. The need for an effective vaccine to curb this pandemic cannot be overemphasized. In view of this, we designed a subcomponent antigenic peptide vaccine targeting the N-terminal (NT) and C-terminal (CT) RNA binding domains of the nucleocapsid protein that aid in viral replication. Promising antigenic B cell and T cell epitopes were predicted using computational pipelines. The peptides "RIRGGDGKMKDL" and "AFGRRGPEQTQGNFG" were the B cell linear epitopes with good antigenic index and nonallergenic property. Two CD8+ and Three CD4+ T cell epitopes were also selected considering their safe immunogenic profiling such as allergenicity, antigen level conservancy, antigenicity, peptide toxicity, and putative restrictions to a number of MHC-I and MHC-II alleles. With these selected epitopes, a nonallergenic chimeric peptide vaccine incapable of inducing a type II hypersensitivity reaction was constructed. The molecular interaction between the Toll-like receptor-5 (TLR5) which was triggered by the vaccine was analyzed by molecular docking and scrutinized using dynamics simulation. Finally, in silico cloning was performed to ensure the expression and translation efficiency of the vaccine, utilizing the pET-28a vector. This research, therefore, provides a guide for experimental investigation and validation.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Proteínas do Nucleocapsídeo/imunologia , Nucleocapsídeo/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/imunologia , Motivos de Ligação ao RNA/imunologia , Receptor 5 Toll-Like/metabolismo , Vacinas Atenuadas/imunologia , Vacinas de Subunidades/imunologia
16.
BMC Bioinformatics ; 21(1): 399, 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907544

RESUMO

BACKGROUND: Cryo-electron tomography is an important and powerful technique to explore the structure, abundance, and location of ultrastructure in a near-native state. It contains detailed information of all macromolecular complexes in a sample cell. However, due to the compact and crowded status, the missing edge effect, and low signal to noise ratio (SNR), it is extremely challenging to recover such information with existing image processing methods. Cryo-electron tomogram simulation is an effective solution to test and optimize the performance of the above image processing methods. The simulated images could be regarded as the labeled data which covers a wide range of macromolecular complexes and ultrastructure. To approximate the crowded cellular environment, it is very important to pack these heterogeneous structures as tightly as possible. Besides, simulating non-deformable and deformable components under a unified framework also need to be achieved. RESULT: In this paper, we proposed a unified framework for simulating crowded cryo-electron tomogram images including non-deformable macromolecular complexes and deformable ultrastructures. A macromolecule was approximated using multiple balls with fixed relative positions to reduce the vacuum volume. A ultrastructure, such as membrane and filament, was approximated using multiple balls with flexible relative positions so that this structure could deform under force field. In the experiment, 400 macromolecules of 20 representative types were packed into simulated cytoplasm by our framework, and numerical verification proved that our method has a smaller volume and higher compression ratio than the baseline single-ball model. We also packed filaments, membranes and macromolecules together, to obtain a simulated cryo-electron tomogram image with deformable structures. The simulated results are closer to the real Cryo-ET, making the analysis more difficult. The DOG particle picking method and the image segmentation method are tested on our simulation data, and the experimental results show that these methods still have much room for improvement. CONCLUSION: The proposed multi-ball model can achieve more crowded packaging results and contains richer elements with different properties to obtain more realistic cryo-electron tomogram simulation. This enables users to simulate cryo-electron tomogram images with non-deformable macromolecular complexes and deformable ultrastructures under a unified framework. To illustrate the advantages of our framework in improving the compression ratio, we calculated the volume of simulated macromolecular under our multi-ball method and traditional single-ball method. We also performed the packing experiment of filaments and membranes to demonstrate the simulation ability of deformable structures. Our method can be used to do a benchmark by generating large labeled cryo-ET dataset and evaluating existing image processing methods. Since the content of the simulated cryo-ET is more complex and crowded compared with previous ones, it will pose a greater challenge to existing image processing methods.


Assuntos
Citoplasma/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Simulação de Dinâmica Molecular , Algoritmos , Análise por Conglomerados , Microscopia Crioeletrônica , Citoplasma/metabolismo , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Razão Sinal-Ruído
17.
Nat Commun ; 11(1): 4734, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948759

RESUMO

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.


Assuntos
Agonistas dos Canais de Cálcio/química , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sítios de Ligação , Microscopia Crioeletrônica , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
18.
PLoS Biol ; 18(9): e3000848, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898131

RESUMO

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Sarcômeros/metabolismo
19.
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
20.
Sci Adv ; 6(37)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32917717

RESUMO

There is an urgent need to repurpose drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent computational-experimental screenings have identified several existing drugs that could serve as effective inhibitors of the virus' main protease, Mpro, which is involved in gene expression and replication. Among these, ebselen (2-phenyl-1,2-benzoselenazol-3-one) appears to be particularly promising. Here, we examine, at a molecular level, the potential of ebselen to decrease Mpro activity. We find that it exhibits a distinct affinity for the catalytic region. Our results reveal a higher-affinity, previously unknown binding site localized between the II and III domains of the protein. A detailed strain analysis indicates that, on such a site, ebselen exerts a pronounced allosteric effect that regulates catalytic site access through surface-loop interactions, thereby inducing a reconfiguration of water hotspots. Together, these findings highlight the promise of ebselen as a repurposed drug against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Azóis/metabolismo , Azóis/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Compostos Organosselênicos/metabolismo , Compostos Organosselênicos/farmacologia , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/metabolismo , Antivirais/metabolismo , Betacoronavirus/metabolismo , Sítios de Ligação , Domínio Catalítico/efeitos dos fármacos , Reposicionamento de Medicamentos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Pandemias , Conformação Proteica/efeitos dos fármacos
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