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1.
Biomolecules ; 11(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34439861

RESUMO

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Burkholderia cepacia/fisiologia , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/fisiologia , Cristalografia por Raios X/métodos , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Virulência/fisiologia
2.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443534

RESUMO

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.


Assuntos
Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Oligoquetos/química , Peptídeo Hidrolases/farmacologia , Trombose/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Bases de Dados de Proteínas , Eritrócitos/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/química , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ativador de Plasminogênio Tecidual/metabolismo , Peixe-Zebra
3.
Int J Mol Sci ; 22(15)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34361054

RESUMO

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Complemento C1q/imunologia , Epitopos/imunologia , Nefrite Lúpica/imunologia , Anticorpos de Cadeia Única/imunologia , Humanos , Nefrite Lúpica/sangue , Estrutura Terciária de Proteína , Subunidades Proteicas
4.
Front Cell Infect Microbiol ; 11: 679982, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235094

RESUMO

Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilis Regulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.


Assuntos
Proteínas de Transporte de Ânions , Genes Bacterianos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Imidazóis , Estrutura Terciária de Proteína , Fator sigma/genética
5.
Methods Enzymol ; 656: 93-122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325801

RESUMO

Recent years have seen a growing number of examples of designed oligomeric molecules with artificial backbone connectivity that are capable of adopting complex folded tertiary structures analogous to those seen in natural proteins. A range of experimental techniques from structural biology and biophysics have been brought to bear in the study of these proteomimetic agents. Here, we discuss some considerations encountered in the characterization of high-resolution folded structure as well as folding thermodynamics of protein-like artificial backbones. We provide an overview of the use of X-ray crystallography and NMR spectroscopy in such systems and review example applications of these methods in the primary literature. Further, we provide detailed protocols for two experiments that have proved useful in our prior and ongoing efforts to compare folding thermodynamics between natural protein domains and heterogeneous-backbone counterparts.


Assuntos
Dobramento de Proteína , Proteínas , Cristalografia por Raios X , Domínios Proteicos , Estrutura Terciária de Proteína , Termodinâmica
6.
Biomolecules ; 11(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201418

RESUMO

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células CHO , Cricetinae , Cricetulus , Glucagon/administração & dosagem , Glucagon/química , Glucagon/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular/métodos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
7.
Biomolecules ; 11(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201823

RESUMO

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.


Assuntos
Aedes/genética , Clonagem Molecular/métodos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Sequência de Aminoácidos , Animais , Feminino , Trato Gastrointestinal/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
8.
Biomolecules ; 11(7)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202543

RESUMO

Adrenergic receptors are G protein-coupled receptors for epinephrine and norepinephrine. They are targets of many drugs for various conditions, including treatment of hypertension, hypotension, and asthma. Adrenergic receptors are intensively studied in structural biology, displayed for binding poses of different types of ligands. Here, we summarized molecular mechanisms of ligand recognition and receptor activation exhibited by structure. We also reviewed recent advances in structure-based ligand discovery against adrenergic receptors.


Assuntos
Agonistas Adrenérgicos/química , Agonistas Adrenérgicos/metabolismo , Antagonistas Adrenérgicos/química , Antagonistas Adrenérgicos/metabolismo , Receptores Adrenérgicos/química , Receptores Adrenérgicos/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X/métodos , Epinefrina/química , Epinefrina/metabolismo , Humanos , Ligantes , Norepinefrina/química , Norepinefrina/metabolismo , Ligação Proteica/fisiologia , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Adrenérgicos/genética
9.
Cell Mol Life Sci ; 78(17-18): 6265-6281, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34241650

RESUMO

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other.


Assuntos
Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Arrestinas/metabolismo , Sítios de Ligação , Quimiocinas/química , Quimiocinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunidade Inata , Microscopia Confocal , Simulação de Acoplamento Molecular , Mutagênese , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética
10.
Cell Mol Life Sci ; 78(17-18): 6305-6318, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34292354

RESUMO

The structural basis for the GTPase-accelerating activity of regulators of G protein signaling (RGS) proteins, as well as the mechanistic basis for their specificity in interacting with the heterotrimeric (αßγ) G proteins they inactivate, is not sufficiently understood at the family level. Here, we used biochemical assays to compare RGS domains across the RGS family and map those individual residues that favorably contribute to GTPase-accelerating activity, and those residues responsible for attenuating RGS domain interactions with Gα subunits. We show that conserved interactions of RGS residues with both the Gα switch I and II regions are crucial for RGS activity, while the reciprocal effects of "modulatory" and "disruptor" residues selectively modulate RGS activity. Our results quantify how specific interactions between RGS domains and Gα subunits are set by a balance between favorable RGS residue interactions with particular Gα switch regions, and unfavorable interactions with the Gα helical domain.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Alinhamento de Sequência , Termodinâmica
11.
Biomed Res Int ; 2021: 9050026, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307671

RESUMO

Chloroflexus aurantiacus is a thermophilic bacterium that produces a multitude of proteins within its genome. Bioinformatics strategies can facilitate comprehending this organism through functional and structural interpretation assessments. This study is aimed at allocating the structure and function through an in silico approach required for bacterial protein biosynthesis. This in silico viewpoint provides copious properties, including the physicochemical properties, subcellular location, three-dimensional structure, protein-protein interactions, and functional elucidation of the protein (WP_012256288.1). The STRING program is utilized for the explication of protein-protein interactions. The in silico investigation documented the protein's hydrophilic nature with predominantly alpha (α) helices in its secondary structure. The tertiary-structure model of the protein has been shown to exhibit reasonably high consistency based on various quality assessment methods. The functional interpretation suggested that the protein can act as a translation initiation factor, a protein required for translation and protein biosynthesis. Protein-protein interactions also demonstrated high credence that the protein interconnected with 30S ribosomal subunit involved in protein synthesis. This study bioinformatically examined that the protein (WP_012256288.1) is affiliated in protein biosynthesis as a translation initiation factor IF-3 of C. aurantiacus.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chloroflexus/metabolismo , Simulação por Computador , Biossíntese de Proteínas , Sequência de Aminoácidos , Domínio Catalítico , Modelos Moleculares , Anotação de Sequência Molecular , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
12.
J Pharmacol Sci ; 147(1): 62-71, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34294374

RESUMO

Owing to the urgent need for therapeutic interventions against the SARS-coronavirus 2 (SARS-CoV-2) pandemic, we employed an in silico approach to evaluate the SARS-CoV-2 inhibitory potential of newly synthesized imidazoles. The inhibitory potentials of the compounds against SARS-CoV-2 drug targets - main protease (Mpro), spike protein (Spro) and RNA-dependent RNA polymerase (RdRp) were investigated through molecular docking analysis. The binding free energy of the protein-ligand complexes were estimated, pharmacophore models were generated and the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the compounds were determined. The compounds displayed various levels of binding affinities for the SARS-CoV-2 drug targets. Bisimidazole C2 scored highest against all the targets, with its aromatic rings including the two imidazole groups contributing to the binding. Among the phenyl-substituted 1H-imidazoles, C9 scored highest against all targets. C11 scored highest against Spro and C12 against Mpro and RdRp among the thiophene-imidazoles. The compounds interacted with HIS 41 - CYS 145 and GLU 288 - ASP 289 - GLU 290 of Mpro, ASN 501 of Spro receptor binding motif and some active site amino acids of RdRp. These novel imidazole compounds could be further developed as drug candidates against SARS-CoV-2 following lead optimization and experimental studies.


Assuntos
Biologia Computacional/métodos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Simulação de Acoplamento Molecular/métodos , SARS-CoV-2/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Imidazóis/química , Imidazóis/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SARS-CoV-2/química , SARS-CoV-2/metabolismo
13.
Bioengineered ; 12(1): 2836-2850, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34227905

RESUMO

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/etiologia , Proteínas de Membrana/genética , SARS-CoV-2 , Serina Endopeptidases/genética , Tupaiidae/genética , Tupaiidae/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Bioengenharia , COVID-19/enzimologia , COVID-19/genética , Biologia Computacional , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Homologia Estrutural de Proteína , Distribuição Tecidual , Tupaiidae/virologia
14.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299054

RESUMO

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/metabolismo , Sítios de Ligação , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Modelos Moleculares , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Ligação Proteica , Estrutura Terciária de Proteína
15.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
16.
Int J Biol Macromol ; 185: 277-286, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34147526

RESUMO

Members of group Bacillus are most widely occurring microbes in agricultural soil and they affect crop health in various ways. They directly stimulate plant growth either by augmenting nutrients availability, invigorating plants' defence mechanisms; repressing soil-borne phytopathogens or by producing growth-regulating hormones like auxins and cytokinins. It is a well known fact that indole-3- acetic acid (a type of auxin) is a vital biologically active phytohormone excreted by certain Bacillus species, but its molecular mechanism has not yet been described. In this study, the auxin efflux carrier gene is isolated from the metagenome of the Tapta Kund hot spring, Uttrakhand, India. In addition, auxin efflux carrier (AEC) transporter protein of Bacillus licheniformis is modeled and the 318 amino acid residues long protein was found homologous to the apical sodium-dependent bile acid transporter (ASBT) of Yersinia frederiksnii, with 10 transmembrane segments (TM1-10) split into different domains: a panel domain defined by TM1, 2, 6 and 7; and a core domain defined by TM3-5 and 8-10. Finally, the predicted Bacillus licheniformis AEC protein has also been phylogenetically evaluated and its detailed molecular transport mechanism was worked out using molecular dynamics simulation analysis. Conclusively, this study demonstrates the efflux mechanism of the substrate, Indole 3- acetic acid by AEC transporter protein.


Assuntos
Bacillus licheniformis/isolamento & purificação , Fontes Termais/microbiologia , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Sequência de Aminoácidos , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Índia , Proteínas de Membrana Transportadoras/metabolismo , Metagenômica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Terciária de Proteína
17.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073028

RESUMO

In the current work we study, via molecular simulations and experiments, the folding and stability of proteins from the tertiary motif of 4-α-helical bundles, a recurrent motif consisting of four amphipathic α-helices packed in a parallel or antiparallel fashion. The focus is on the role of the loop region in the structure and the properties of the wild-type Rop (wtRop) and RM6 proteins, exploring the key factors which can affect them, through all-atom molecular dynamics (MD) simulations and supporting by experimental findings. A detailed investigation of structural and conformational properties of wtRop and its RM6 loopless mutation is presented, which display different physical characteristics even in their native states. Then, the thermal stability of both proteins is explored showing RM6 as more thermostable than wtRop through all studied measures. Deviations from native structures are detected mostly in tails and loop regions and most flexible residues are indicated. Decrease of hydrogen bonds with the increase of temperature is observed, as well as reduction of hydrophobic contacts in both proteins. Experimental data from circular dichroism spectroscopy (CD), are also presented, highlighting the effect of temperature on the structural integrity of wtRop and RM6. The central goal of this study is to explore on the atomic level how a protein mutation can cause major changes in its physical properties, like its structural stability.


Assuntos
Proteínas de Bactérias/química , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Ligação de Hidrogênio , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura
18.
Emerg Microbes Infect ; 10(1): 1293-1299, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34125658

RESUMO

The SARS-CoV-2 B.1.1.7 lineage is highly infectious and as of April 2021 accounted for 92% of COVID-19 cases in Europe and 59% of COVID-19 cases in the U.S. It is defined by the N501Y mutation in the receptor-binding domain (RBD) of the Spike (S) protein, and a few other mutations. These include two mutations in the N terminal domain (NTD) of the S protein, HV69-70del and Y144del (also known as Y145del due to the presence of tyrosine at both positions). We recently identified several emerging SARS-CoV-2 variants of concerns, characterized by Membrane (M) protein mutations, including I82T and V70L. We now identify a sub-lineage of B.1.1.7 that emerged through sequential acquisitions of M:V70L in November 2020 followed by a novel S:D178H mutation first observed in early February 2021. The percentage of B.1.1.7 isolates in the US that belong to this sub-lineage increased from 0.15% in February 2021 to 1.8% in April 2021. To date, this sub-lineage appears to be U.S.-specific with reported cases in 31 states, including Hawaii. As of April 2021, it constituted 36.8% of all B.1.1.7 isolates in Washington. Phylogenetic analysis and transmission inference with Nextstrain suggest this sub-lineage likely originated in either California or Washington. Structural analysis revealed that the S:D178H mutation is in the NTD of the S protein and close to two other signature mutations of B.1.1.7, HV69-70del and Y144del. It is surface exposed and may alter NTD tertiary configuration or accessibility, and thus has the potential to affect neutralization by NTD directed antibodies.


Assuntos
Mutação , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Sequenciamento Completo do Genoma/métodos , Sítios de Ligação , Humanos , Modelos Moleculares , Filogenia , Domínios Proteicos , Estrutura Terciária de Proteína , SARS-CoV-2/genética , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Estados Unidos
19.
Front Immunol ; 12: 612807, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34163462

RESUMO

Since being identified as a key receptor for SARS-CoV-2, Angiotensin converting enzyme 2 (ACE2) has been studied as one of the potential targets for the development of preventative and/or treatment options. Tissue expression of ACE2 and the amino acids interacting with the spike protein of SARS-CoV-2 have been mapped. Furthermore, the recombinant soluble extracellular domain of ACE2 is already in phase 2 trials as a treatment for SARS-CoV-2 infection. Most studies have continued to focus on the ACE2 extracellular domain, which is known to play key roles in the renin angiotensin system and in amino acid uptake. However, few also found ACE2 to have an immune-modulatory function and its intracellular tail may be one of the signaling molecules in regulating cellular activation. The implication of its immune-modulatory role in preventing the cytokine-storm, observed in severe COVID-19 disease outcomes requires further investigation. This review focuses on the regulated proteolytic cleavage of ACE2 upon binding to inducer(s), such as the spike protein of SARS-CoV, the potential of cleaved ACE2 intracellular subdomain in regulating cellular function, and the ACE2's immune-modulatory function. This knowledge is critical for targeting ACE2 levels for developing prophylactic treatment or preventative measures in SARS-CoV infections.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Membrana Celular/metabolismo , SARS-CoV-2/patogenicidade , Enzima de Conversão de Angiotensina 2/química , COVID-19/metabolismo , COVID-19/virologia , Humanos , Imunomodulação , Estrutura Terciária de Proteína , Proteólise , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
20.
Nat Commun ; 12(1): 3921, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168113

RESUMO

We previously elucidated principles for designing ideal proteins with completely consistent local and non-local interactions which have enabled the design of a wide range of new αß-proteins with four or fewer ß-strands. The principles relate local backbone structures to supersecondary-structure packing arrangements of α-helices and ß-strands. Here, we test the generality of the principles by employing them to design larger proteins with five- and six- stranded ß-sheets flanked by α-helices. The initial designs were monomeric in solution with high thermal stability, and the nuclear magnetic resonance (NMR) structure of one was close to the design model, but for two others the order of strands in the ß-sheet was swapped. Investigation into the origins of this strand swapping suggested that the global structures of the design models were more strained than the NMR structures. We incorporated explicit consideration of global backbone strain into the design methodology, and succeeded in designing proteins with the intended unswapped strand arrangements. These results illustrate the value of experimental structure determination in guiding improvement of de novo design, and the importance of consistency between local, supersecondary, and global tertiary interactions in determining protein topology. The augmented set of principles should inform the design of larger functional proteins.


Assuntos
Engenharia de Proteínas/métodos , Proteínas/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/genética
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