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1.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064883

RESUMO

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases-a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.


Assuntos
Amiloide/química , Proteínas Priônicas/química , Multimerização Proteica , Temperatura , Conformação Proteica
2.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34065004

RESUMO

Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides make them an ideal candidate for metal-related protease inhibitors. This research investigates the influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides. The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the desired compounds is conveniently available from the three-component condensation of appropriate amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is the careful selection of the protecting groups for all the functionals. Determination of the inhibitor activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient procedure for the preparation of phosphinotripeptides has been performed. Activity test shown that introduction of additional residue into P2 position obtains the micromolar range inhibitors of SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its selectivity toward mammalian and plant leucyl aminopeptidases.


Assuntos
Inibidores Enzimáticos/farmacologia , Leucil Aminopeptidase/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Fosfinas/química , Animais , Inibidores Enzimáticos/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Suínos
3.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34065025

RESUMO

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Ciclobutanos/metabolismo , Leishmania/metabolismo , Peptidomiméticos/metabolismo , Prolina/metabolismo , Sobrevivência Celular , Peptídeos Penetradores de Células/química , Ciclobutanos/química , Dimerização , Células HeLa , Humanos , Peptidomiméticos/química , Prolina/química , Conformação Proteica
4.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066237

RESUMO

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.


Assuntos
Amiloide/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Agregados Proteicos , Conformação Proteica , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Homologia de Sequência
5.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068366

RESUMO

Magnaporthe oryzae (M. oryzae) is a typical cause of rice blast in agricultural production. Isobavachalcone (IBC), an active ingredient of Psoralea corylifolia L. extract, is an effective fungicide against rice blast. To determine the mechanism of IBC against M. oryzae, the effect of IBC on the metabolic pathway of M. oryzae was explored by transcriptome profiling. In M. oryzae, the expression of pyruvate dehydrogenase E1 (PDHE1), part of the tricarboxylic acid (TCA cycle), was significantly decreased in response to treatment with IBC, which was verified by qPCR and testing of enzyme activity. To further elucidate the interactions between IBC and PDHE1, the 3D structure model of the PDHE1 from M. oryzae was established based on homology modeling. The model was utilized to analyze the molecular interactions through molecular docking and molecular dynamics simulation, revealing that IBC has π-π stacking interactions with residue TYR139 and undergoes hydrogen bonding with residue ASP217 of PDHE1. Additionally, the nonpolar residues PHE111, MET174, ILE 187, VAL188, and MET250 form strong hydrophobic interactions with IBC. The above results reveal that PDHE1 is a potential target for antifungal agents, which will be of great significance for guiding the design of new fungicides. This research clarified the mechanism of IBC against M. oryzae at the molecular level, which will underpin further studies of the inhibitory mechanism of flavonoids and the discovery of new targets. It also provides theoretical guidance for the field application of IBC.


Assuntos
Chalconas/farmacologia , Proteínas Fúngicas/metabolismo , Magnaporthe/efeitos dos fármacos , Oryza/enzimologia , Doenças das Plantas/imunologia , Piruvato Desidrogenase (Lipoamida)/antagonistas & inibidores , Transcriptoma/efeitos dos fármacos , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Magnaporthe/fisiologia , Simulação de Acoplamento Molecular , Oryza/efeitos dos fármacos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Conformação Proteica , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068417

RESUMO

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.


Assuntos
Ataxia/patologia , Canais de Cálcio/genética , Mutação , Adulto , Sequência de Aminoácidos , Ataxia/congênito , Ataxia/etiologia , Ataxia/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Criança , Feminino , Humanos , Masculino , Neuroimagem , Fenótipo , Conformação Proteica , Homologia de Sequência , Relação Estrutura-Atividade , Adulto Jovem
7.
Viruses ; 13(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071984

RESUMO

The high sequence identity of the first SARS-CoV-2 samples collected in December 2019 at Wuhan did not foretell the emergence of novel variants in the United Kingdom, North and South America, India, or South Africa that drive the current waves of the pandemic. The viral spike receptor possesses two surface areas of high mutagenic plasticity: the supersite in its N-terminal domain (NTD) that is recognised by all anti-NTD antibodies and its receptor binding domain (RBD) where 17 residues make contact with the human Ace2 protein (angiotensin I converting enzyme 2) and many neutralising antibodies bind. While NTD mutations appear at first glance very diverse, they converge on the structure of the supersite. The mutations within the RBD, on the other hand, hone in on only a small number of key sites (K417, L452, E484, N501) that are allosteric control points enabling spike to escape neutralising antibodies while maintaining or even gaining Ace2-binding activity. The D614G mutation is the hallmark of all variants, as it promotes viral spread by increasing the number of open spike protomers in the homo-trimeric receptor complex. This review discusses the recent spike mutations as well as their evolution.


Assuntos
COVID-19/virologia , Variação Genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sítios de Ligação/genética , COVID-19/transmissão , Evolução Molecular , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química
8.
Viruses ; 13(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072569

RESUMO

The COVID-19 pandemic, which began in Wuhan (Hubei, China), has been ongoing for about a year and a half. An unprecedented number of people around the world have been infected with SARS-CoV-2, the etiological agent of COVID-19. Despite the fact that the mortality rate for COVID-19 is relatively low, the total number of deaths has currently already reached more than three million and continues to increase due to high incidence. Since the beginning of the pandemic, a large number of sequences have been obtained and many genetic variants have been identified. Some of them bear significant mutations that affect biological properties of the virus. These genetic variants, currently Variants of Concern (VoC), include the so-called United Kingdom variant (20I/501Y), the Brazilian variant (20J/501Y.V3), and the South African variant (20H/501Y.V2). We describe here a novel SARS-CoV-2 variant with distinct spike protein mutations, first obtained at the end of January 2021 in northwest Russia. Therefore, it is necessary to pay attention to the dynamics of its spread among patients with COVID-19, as well as to study in detail its biological properties.


Assuntos
SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Análise Mutacional de DNA , DNA Complementar , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Conformação Proteica , Federação Russa , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química
9.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073705

RESUMO

It is essential for future research to develop a new, reliable prediction method of DNA binding sites because DNA binding sites on DNA-binding proteins provide critical clues about protein function and drug discovery. However, the current prediction methods of DNA binding sites have relatively poor accuracy. Using 3D coordinates and the atom-type of surface protein atom as the input, we trained and tested a deep learning model to predict how likely a voxel on the protein surface is to be a DNA-binding site. Based on three different evaluation datasets, the results show that our model not only outperforms several previous methods on two commonly used datasets, but also demonstrates its robust performance to be consistent among the three datasets. The visualized prediction outcomes show that the binding sites are also mostly located in correct regions. We successfully built a deep learning model to predict the DNA binding sites on target proteins. It demonstrates that 3D protein structures plus atom-type information on protein surfaces can be used to predict the potential binding sites on a protein. This approach should be further extended to develop the binding sites of other important biological molecules.


Assuntos
Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Aprendizado Profundo , Software , Animais , Sítios de Ligação , Humanos , Conformação Proteica , Proteoma
10.
Science ; 372(6546)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083463

RESUMO

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno de Histocompatibilidade H-2D/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T , Feminino , Antígeno de Histocompatibilidade H-2D/química , Antígeno de Histocompatibilidade H-2D/imunologia , Vírus da Influenza A , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais
11.
Viruses ; 13(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067878

RESUMO

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19/virologia , Polissacarídeos/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Sítios de Ligação , Glicosilação , Interações entre Hospedeiro e Microrganismos , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ligação Viral
12.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070174

RESUMO

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.


Assuntos
Endopeptidase Clp/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Bactérias/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/etiologia , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/fisiologia , Zoonoses Bacterianas/etiologia , Endopeptidase Clp/química , Proteínas de Choque Térmico/fisiologia , Humanos , Modelos Moleculares , Conformação Proteica , Virulência/fisiologia
13.
Nat Commun ; 12(1): 3305, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083522

RESUMO

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química
14.
Nat Commun ; 12(1): 3427, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103518

RESUMO

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.


Assuntos
Peptídeos/química , Peptídeos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Determinação de Ponto Final , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Oleicos/química , Peptídeos/farmacologia , Placebos , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Termodinâmica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
15.
Chem Commun (Camb) ; 57(43): 5314-5317, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-33942835

RESUMO

While the state-of-the-art computational simulations support the neutral state for the catalytic dyad of the SARS-CoV-2 main protease, the recently-reported neutron structure exhibits a zwitterionic form. To better compare the structural and dynamical features of the two charge configurations, we perform a Molecular Dynamics study of the dimeric enzyme in complex with a peptide substrate. The simulations show that the enzyme charge configuration from the neutron structure is not compatible with a catalytically-competent binding mode for peptide substrates.


Assuntos
Peptídeo Hidrolases/química , SARS-CoV-2/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Peptídeos , Conformação Proteica
16.
Nat Commun ; 12(1): 2694, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976221

RESUMO

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.


Assuntos
Conformação Proteica , Multimerização Proteica , Receptores de N-Metil-D-Aspartato/química , Regulação Alostérica , Transferência Ressonante de Energia de Fluorescência/métodos , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Células HEK293 , Humanos , Cinética , Microscopia Confocal , Modelos Moleculares , Ligação Proteica , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
17.
J Phys Chem Lett ; 12(20): 4814-4822, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-33999630

RESUMO

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , COVID-19/tratamento farmacológico , Imidazóis/química , Leucina/análogos & derivados , Inibidores de Proteases/química , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/metabolismo , Domínio Catalítico , Desenho de Fármacos , Humanos , Imidazóis/farmacologia , Leucina/química , Leucina/farmacologia , Ligantes , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , SARS-CoV-2/metabolismo
18.
J Vis Exp ; (170)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33938889

RESUMO

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the µs-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a 15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the µs-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica , Cinética , Isótopos de Nitrogênio , Termodinâmica
19.
Chem Asian J ; 16(12): 1634-1642, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-33949124

RESUMO

Computational drug design is increasingly becoming important with new and unforeseen diseases like COVID-19. In this study, we present a new computational de novo drug design and repurposing method and applied it to find plausible drug candidates for the receptor binding domain (RBD) of SARS-CoV-2 (COVID-19). Our study comprises three steps: atom-by-atom generation of new molecules around a receptor, structural similarity mapping to existing approved and investigational drugs, and validation of their binding strengths to the viral spike proteins based on rigorous all-atom, explicit-water well-tempered metadynamics free energy calculations. By choosing the receptor binding domain of the viral spike protein, we showed that some of our new molecules and some of the repurposable drugs have stronger binding to RBD than hACE2. To validate our approach, we also calculated the free energy of hACE2 and RBD, and found it to be in an excellent agreement with experiments. These pool of drugs will allow strategic repurposing against COVID-19 for a particular prevailing conditions.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Desenho de Fármacos , Reposicionamento de Medicamentos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Antivirais/química , COVID-19/virologia , Simulação por Computador , Humanos , Modelos Moleculares , Conformação Proteica , Termodinâmica
20.
Emerg Microbes Infect ; 10(1): 1016-1023, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34003073

RESUMO

Given the on-going SARS-CoV-2 pandemic, identification of immunogenic targets against the viral protein will provide crucial advances towards the development of sensitive diagnostic tools and vaccination strategies. Our previous study has found that ORF8 protein of SARS-CoV-2 is highly immunogenic and shows high sensitivity in identifying COVID-19 disease. In this study, by employing overlapping linear peptides, we characterized the IgG immunodominant regions on SARS-CoV-2 ORF8 protein that are seropositive in the sera from SARS-CoV-2-infected patients. The major immunogenic epitopes are localized at (1) N-termini alpha helix, (2) the resides spanning beta 2 and 3 sheets, and (3) the loop between beta 4 and 5 sheets. Additionally, hamster model infected by SARS-CoV-2 further validates the seropositivity of the linear epitopes in vivo, demonstrating a potential application of the linear peptide-based immunization strategy. Taken together, identification and validation of these B-cell linear epitopes will provide insights into the design of serological diagnostics and peptide-based vaccination approach against this pandemic virus of high priority.


Assuntos
COVID-19/imunologia , Epitopos de Linfócito B , SARS-CoV-2/imunologia , Proteínas Virais/química , Animais , Anticorpos Antivirais , Cricetinae , Humanos , Epitopos Imunodominantes , Mesocricetus , Modelos Moleculares , Conformação Proteica , Proteínas Virais/imunologia
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