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1.
Nature ; 574(7779): 581-585, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645725

RESUMO

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Animais , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P2Y1/química , Transdução de Sinais , Anticorpos de Domínio Único/química , Especificidade da Espécie , Ácido Succínico/metabolismo
2.
Life Sci ; 233: 116710, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369762

RESUMO

AIMS: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors. MATERIALS AND METHODS: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging. KEY FINDINGS: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case. SIGNIFICANCE: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors.


Assuntos
Apoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Sistemas de Liberação de Medicamentos , Mioglobina/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Apoproteínas/química , Apoptose/efeitos da radiação , Bovinos , Sobrevivência Celular , Curcumina/química , Células HeLa , Cavalos , Humanos , Mioglobina/química , Fármacos Fotossensibilizantes/química , Soroalbumina Bovina/química
3.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
4.
Nature ; 567(7748): 409-413, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30867599

RESUMO

Chromatin remodellers include diverse enzymes with distinct biological functions, but nucleosome-sliding activity appears to be a common theme1,2. Among the remodelling enzymes, Snf2 serves as the prototype to study the action of this protein family. Snf2 and related enzymes share two conserved RecA-like lobes3, which by themselves are able to couple ATP hydrolysis to chromatin remodelling. The mechanism by which these enzymes couple ATP hydrolysis to translocate the nucleosome along the DNA remains unclear2,4-8. Here we report the structures of Saccharomyces cerevisiae Snf2 bound to the nucleosome in the presence of ADP and ADP-BeFx. Snf2 in the ADP-bound state adopts an open conformation similar to that in the apo state, and induces a one-base-pair DNA bulge at superhelix location 2 (SHL2), with the tracking strand showing greater distortion than the guide strand. The DNA distortion propagates to the proximal end, leading to staggered translocation of the two strands. The binding of ADP-BeFx triggers a closed conformation of the enzyme, resetting the nucleosome to a relaxed state. Snf2 shows altered interactions with the DNA in different nucleotide states, providing the structural basis for DNA translocation. Together, our findings suggest a fundamental mechanism for the DNA translocation that underlies chromatin remodelling.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Apoproteínas/química , Apoproteínas/metabolismo , Transporte Biológico , Cromatina/química , DNA/química , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
5.
Nature ; 567(7748): 389-393, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842659

RESUMO

Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS)1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses.


Assuntos
Galinhas , Microscopia Crioeletrônica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Nucleotídeos Cíclicos/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Nucleotídeos Cíclicos/química
6.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 2): 98-104, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30713160

RESUMO

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRα, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π-bulge in helix H7, which was originally built as a regular α-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1-H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRα LBD.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos
7.
Hum Cell ; 32(2): 103-113, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30635859

RESUMO

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.


Assuntos
Pulmão/embriologia , Organoides , Alvéolos Pulmonares , Mucosa Respiratória/crescimento & desenvolvimento , Animais , Apoproteínas/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Colágeno/farmacologia , Meios de Cultura/farmacologia , Combinação de Medicamentos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/farmacologia , Laminina/farmacologia , Camundongos Endogâmicos ICR , Proteoglicanas/farmacologia , Alvéolos Pulmonares/citologia , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Selênio/farmacologia , Estimulação Química , Transferrina/farmacologia
8.
Food Chem ; 278: 388-395, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30583389

RESUMO

α-Lactalbumin (ALA) is a Ca2+-binding protein which constitutes up to 20% of whey protein. At acidic pH, and in the apo-state at elevated temperatures, ALA is the classic 'molten globule' (MG). This study examined epigallocatechin-3-gallate (EGCG) binding to ALA in its apo form (apoALA) and stabilizing effect on protein structure thereof. EGCG binds to apoALA in both native and MG state. The complex of EGCG and ALA is more stable to thermal denaturation. The docking analysis and molecular dynamic simulation (MDS) showed that Ca2+ removal decreased conformational stability of ALA, because of the local destabilization of Ca2+-binding region. EGCG binding to apoALA increases its stability by reverting of conformation and stability of Ca2+-binding region. Therefore, EGCG-induced thermal stability of apoALA is based on increased apoALA conformational rigidity. This study implies that during gastric digestion of tea with milk EGCG would remain bound to ALA, albeit in the Ca2+-free form.


Assuntos
Apoproteínas/química , Catequina/análogos & derivados , Lactalbumina/química , Simulação de Dinâmica Molecular , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Cálcio/química , Catequina/química , Catequina/metabolismo , Concentração de Íons de Hidrogênio , Lactalbumina/metabolismo , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência
9.
J Chem Inf Model ; 59(1): 597-604, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30525607

RESUMO

Allosteric modulators, by targeting the less-conserved allosteric sites, represent an innovative strategy in drug discovery. These modulators have a distinctive advantage over orthosteric ligands that attach to the conserved, functional orthosteric sites. However, in structure-based drug design, it remains unclear whether allosteric protein structures determined without orthosteric ligand binding are suitable for allosteric drug screening. In this study, we performed large-scale conformational samplings of six representative allosteric proteins uncomplexed ( apo) and complexed ( holo) with orthosteric ligands to explore the effect of orthosteric site binding on the conformational dynamics of allosteric sites. The results, coupled with the redocking evaluation of allosteric modulators to their apo and holo proteins using their MD trajectories, indicated that orthosteric site binding had an effect on the dynamics of the allosteric sites and allosteric modulators preferentially bound to their holo proteins. According to the analysis data, we constructed a new correlation model for quantifying the allosteric site change driven by substrate binding to the orthosteric site. These results highlight the strong demand to select holo allosteric proteins as initial inputs in structure-based allosteric drug screening when the distance between orthosteric and allosteric sites in the protein is below 5 Å, which is expected to contribute to allosteric drug discovery.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Descoberta de Drogas/métodos , Simulação de Dinâmica Molecular , Sítio Alostérico/efeitos dos fármacos , Humanos , Conformação Proteica
10.
Proc Natl Acad Sci U S A ; 115(39): E9085-E9094, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30201724

RESUMO

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.


Assuntos
Apoproteínas/química , Chaetomium/química , Proteínas Fúngicas/química , Proteínas de Ligação ao GTP/química , Proteína 1 Reguladora do Ferro/química , Proteínas com Ferro-Enxofre/química , Motivos de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Domínio Catalítico , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo
11.
Nature ; 556(7700): 203-208, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618818

RESUMO

The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 ß-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between ß-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc 1 complex.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Hyphomicrobiaceae/química , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/ultraestrutura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Benzoquinonas/metabolismo , Sítios de Ligação , Complexos de Proteínas Captadores de Luz/metabolismo , Magnésio/química , Magnésio/metabolismo , Modelos Moleculares , Fotossíntese , Conformação Proteica , Estabilidade Proteica
12.
Drug Metab Dispos ; 46(6): 813-822, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29602797

RESUMO

17α-Ethynylestradiol (EE), a major component of many oral contraceptives, affects the activities of a number of the human cytochrome P450 (P450) enzymes. Here, we characterized the effect of EE on CYP2J2, a major human P450 isoform that participates in metabolism of arachidonic acid. EE inactivated the hydroxyebastine carboxylation activity of CYP2J2 in a reconstituted system. The loss of activity is time and concentration dependent and requires NADPH. The KI and kinact values for the inactivation were 3.6 µM and 0.08 minute-1, respectively. Inactivation of CYP2J2 by EE was due to formation of a heme adduct as well as an apoprotein adduct. Mass spectral analysis of CYP2J2 partially inactivated by EE showed two distinct protein masses in the deconvoluted spectrum that exhibited a mass difference of approximately 312 Da, which is equivalent to the sum of the mass of EE and one oxygen atom. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a heme adduct with MH+ ion at m/z 875.5, corresponding to alkylation of an iron-depleted prosthetic heme by EE plus one oxygen atom. The reactive intermediate responsible for covalently modifying both the prosthetic heme and apoprotein was characterized by trapping with glutathione (GSH). LC-MS/MS analysis revealed two GSH conjugate isomers with MH+ ions at m/z 620, which were formed by reaction between GSH and EE with the oxygen being added to either the internal or terminal carbon of the ethynyl moiety. High-pressure liquid chromatography analysis revealed that three other major metabolites were formed during EE metabolism by CYP2J2.


Assuntos
Apoproteínas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Etinilestradiol/farmacologia , Heme/metabolismo , Ácido Araquidônico/metabolismo , Glutationa/metabolismo , Humanos , NADP/metabolismo , Oxigênio/metabolismo
13.
PLoS Comput Biol ; 14(4): e1006072, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29614072

RESUMO

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.


Assuntos
Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Sinalização do Cálcio , Biologia Computacional , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Eletricidade Estática
14.
Nature ; 556(7699): 122-125, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29512653

RESUMO

The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S2. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported, but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α-subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the binding interactions at S1 and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative subdomain orientations and triggers downstream signal propagation.


Assuntos
Microscopia Crioeletrônica , Insulina/química , Insulina/metabolismo , Multimerização Proteica , Receptor de Insulina/química , Receptor de Insulina/ultraestrutura , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Receptor de Insulina/metabolismo , Transdução de Sinais , Imagem Individual de Molécula
15.
Int J Mol Sci ; 19(1)2018 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-29337899

RESUMO

Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.


Assuntos
Ceruloplasmina/metabolismo , Cobre/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Apoproteínas/metabolismo , Estabilidade Enzimática , Proteínas Mutantes/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência , Ureia/farmacologia
16.
J Biomol Struct Dyn ; 36(1): 221-232, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28024445

RESUMO

Structures of many metal-binding proteins are often obtained without structural cations in their apoprotein forms. Missing cation coordinates are usually updated from structural templates constructed from many holoprotein structures. Such templates usually do not include structural water, the important contributor to the ion binding energy. Structural templates are also inconvenient for taking into account structural modifications around the binding site at apo-/holo- transitions. An approach based upon statistical potentials readily takes into account structural modifications associated with binding as well as contribution of structural water molecules. Here, we construct a set of statistical potentials for Mg2+, Ca2+, and Zn2+ contacting with protein atoms of a different type or structural water oxygens. Each type of the cations tends to form tight contacts with protein atoms of specific types. Structural water contributes relatively more into the binding pseudo-energy of Mg2+ and Ca2+ than of Zn2+. We have developed PIONCA (Protein-Ion Calculator), a fast CUDA GPGPU-based algorithm that predicts ion-binding sites in apoproteins. Comparative tests demonstrate that PIONCA outperforms most of the tools based on structural templates or docking. Our software can be also used for locating bound cations in holoprotein structures with missing cation heteroatoms. PIONCA is equipped with an interactive web interface based upon JSmol.


Assuntos
Apoproteínas/química , Cátions/química , Metais/química , Água/química , Algoritmos , Apoproteínas/metabolismo , Sítios de Ligação , Cátions/metabolismo , Biologia Computacional/métodos , Metais/metabolismo , Ligação Proteica , Software , Termodinâmica
17.
Int J Biol Macromol ; 107(Pt A): 626-634, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28919529

RESUMO

Post-translational modifications play important roles in conformational properties and aggregation propensities of different peptides and proteins. In the present study, we have investigated the effects of acetylation of lysine residues on the structure and aggregation properties of apomyoglobin (apoMb).All of the 19 lysine residues were modified. Far-, near-UV CD, intrinsic and acrylamide quenching fluorescence studies indicated that acetylation significantly influences conformation of apoMb by altering both its secondary and tertiary structures. A considerable decrease in ANS fluorescence intensity was observed, which also suggested disruption of the heme pocket. Dynamic light scattering indicated partial compaction of protein structure as a consequence of the shielding effect of acetylation. While the presence of well-defined mature fibrils was detected in solutions of native apoMb, acetylation promoted formation of non-toxic amorphous aggregates, with low ß-sheets content and decreased affinity for Thioflavin T, an amyloid-specific dye. Results are discussed in terms of the role of surface charge in conformational alterations of proteins and how small changes in ionic networks may affect aggregation pathways and morphology of the resulting aggregates. The physiological significance of the modification process in controlling cytotoxicity of the aggregated species is also discussed.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Apoproteínas/metabolismo , Heme/metabolismo , Lisina/metabolismo , Mioglobina/metabolismo , Agregados Proteicos , Processamento de Proteína Pós-Traducional , Acetilação , Proteínas Amiloidogênicas/química , Animais , Apoproteínas/química , Sobrevivência Celular/efeitos dos fármacos , Heme/química , Cavalos , Lisina/química , Mioglobina/química , Células PC12 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Espectrometria de Fluorescência , Eletricidade Estática , Tiazóis/química
18.
Photosynth Res ; 135(1-3): 125-139, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28236074

RESUMO

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.


Assuntos
Apoproteínas/metabolismo , Proteínas de Bactérias/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Apoproteínas/química , Proteínas de Bactérias/química , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cisteína/metabolismo , Difusão , Hidrodinâmica , Espectrometria de Massas , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Coloração e Rotulagem , Compostos de Sulfidrila/metabolismo
19.
Structure ; 26(1): 153-160.e4, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29276033

RESUMO

BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.


Assuntos
Antineoplásicos/química , Apoproteínas/química , Antígenos de Histocompatibilidade Menor/química , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Antineoplásicos/síntese química , Apoproteínas/antagonistas & inibidores , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Biophys J ; 113(12): 2805-2814, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29262373

RESUMO

UnaG is a recently discovered ligand-induced fluorescent protein that utilizes bound bilirubin (BR) as its fluorophore. The fluorescence of the UnaG-BR complex (holoUnaG) compares in quantum efficiency to that of enhanced green fluorescent protein, but it is superior in that the fluorophore formation is instantaneous and not dependent on oxygen; hence, much attention has been paid to UnaG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as the association/dissociation rates of BR, which determine the stability thereof, and the dispersibility of UnaG in aqueous solutions, which influence the functions of labeled proteins. In this study, we found, in the process of investigating the association rate, that the holoUnaG takes two distinct fluorescence states, which we named holoUnaG1 and holoUnaG2. The holoUnaG1 initially forms after binding BR and then changes to the brighter holoUnaG2 by a reversible intra-molecular reaction, thereby finally reaching an equilibrium between the two states. Spectroscopic analysis indicated that the intra-molecular reaction was associated not with a chemical change of BR but with a change in the environmental conditions surrounding BR. We also revealed that the molecular brightness ratio and equilibrium population ratio of the two states (holoUnaG1/holoUnaG2) were 1:3.9 and 6:4, respectively, using photon number counting analysis. From these results, we have suggested a novel schema, to our knowledge, for the formation of the UnaG and BR complex system and have determined the various rate constants associated therein. Additionally, using analytical ultracentrifugation, we established that UnaG in the apo-state (apoUnaG) and the holoUnaG are monomeric in aqueous solution. These findings provide not only key information for the practical use of UnaG as a fluorescent probe, but also the possibility for development of a brighter UnaG mutant by genetic engineering to constitutive holoUnaG2.


Assuntos
Bilirrubina/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Relação Dose-Resposta a Droga , Ligantes , Cloreto de Sódio/farmacologia , Espectrometria de Fluorescência , Água/química
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