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1.
J Agric Food Chem ; 67(42): 11796-11804, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31589426

RESUMO

High molecular weight glutenin subunits (HMW-GSs) are important components of wheat (Triticum aestivum L.) gluten proteins that affect the processing quality of wheat dough. However, the contributions of individual HMW-GS to sponge cake quality still remain unclear. To investigate the effects of 1Bx7 and 1By9 at Glu-B1 on wheat gluten properties and sponge cake quality, we screened an ethylmethanesulfonate (EMS)-mutagenized wheat population and obtained two independent mutants that lack 1Bx7 and another two mutants that lack 1By9. The absence of 1Bx7 or 1By9 significantly affects the accumulation levels of gluten proteins and the formation of a gluten network. Quality testing indicated that the lack of 1Bx7 or 1By9 leads to weaker dough strength and inferior sponge cake performance. These results demonstrate that 1Bx7 and 1By9 make important contributions to gluten functionality and sponge cake quality.


Assuntos
Pão/análise , Glutens/química , Triticum/química , Culinária , Glutens/genética , Peso Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Triticum/genética
2.
Nature ; 574(7777): 278-282, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578520

RESUMO

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.


Assuntos
Proteína Centromérica A/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
3.
BMC Bioinformatics ; 20(1): 464, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500562

RESUMO

BACKGROUND: The function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment. RESULTS: We present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function. CONCLUSIONS: Our findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.


Assuntos
Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Complexos Multiproteicos/química , Multimerização Proteica , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Solventes
4.
Cell Mol Life Sci ; 76(20): 4023-4042, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31236625

RESUMO

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.


Assuntos
Mitocôndrias/enzimologia , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Subunidades Proteicas/química , Proteínas/genética , Succinato Desidrogenase/química , Animais , Ciclo do Ácido Cítrico/genética , Coenzimas/química , Coenzimas/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fosforilação Oxidativa , Plantas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
5.
Biochemistry (Mosc) ; 84(4): 407-415, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228932

RESUMO

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues ß139, ß158, ß189, and ß319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The ßN158L substitution produced no effect on the enzyme activity, while substitutions ßF139Y, ßF189L, and ßV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations ßF189L and ßV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues ß139, ß189, and ß319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Força Próton-Motriz , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Alinhamento de Sequência
6.
J Korean Med Sci ; 34(20): e149, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31124326

RESUMO

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.


Assuntos
Clonorchis sinensis/genética , DNA Antigo/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Oxirredutases/genética , Animais , Clonorquíase/diagnóstico , Clonorquíase/epidemiologia , Clonorchis sinensis/classificação , DNA Antigo/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/classificação , Humanos , Múmias/parasitologia , Oxirredutases/química , Oxirredutases/classificação , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/classificação , Subunidades Proteicas/genética , República da Coreia , Análise de Sequência de DNA
7.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 359-367, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045565

RESUMO

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Šresolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Mycobacterium tuberculosis/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Divisão Celular , Clonagem Molecular , Cristalografia por Raios X , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Temperatura Ambiente
8.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934865

RESUMO

One of the known potential effects of disease-causing amino acid substitutions in proteins is to modulate protein-protein interactions (PPIs). To interpret such variants at the molecular level and to obtain useful information for prediction purposes, it is important to determine whether they are located at protein-protein interfaces, which are composed of two main regions, core and rim, with different evolutionary conservation and physicochemical properties. Here we have performed a structural, energetics and computational analysis of interactions between proteins hosting mutations related to diseases detected in newborn screening. Interface residues were classified as core or rim, showing that the core residues contribute the most to the binding free energy of the PPI. Disease-causing variants are more likely to occur at the interface core region rather than at the interface rim (p < 0.0001). In contrast, neutral variants are more often found at the interface rim or at the non-interacting surface rather than at the interface core region. We also found that arginine, tryptophan, and tyrosine are over-represented among mutated residues leading to disease. These results can enhance our understanding of disease at molecular level and thus contribute towards personalized medicine by helping clinicians to provide adequate diagnosis and treatments.


Assuntos
Biologia Computacional/métodos , Doença/genética , Mutação/genética , Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Humanos , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Doenças do Recém-Nascido/genética , Simulação de Acoplamento Molecular , Proteínas Mutantes/química , Triagem Neonatal , Ligação Proteica , Subunidades Proteicas/química , Globinas beta/química
9.
Nat Commun ; 10(1): 1520, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944318

RESUMO

In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB-OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Membrana Transportadoras/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade
10.
Science ; 364(6438): 355-362, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30975770

RESUMO

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1-GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo-electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.


Assuntos
Receptores de AMPA/química , Animais , Encéfalo/metabolismo , Microscopia Crioeletrônica , Ativação do Canal Iônico , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Ratos , Receptores de AMPA/genética , Imagem Individual de Molécula
11.
Nat Commun ; 10(1): 1740, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988355

RESUMO

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.


Assuntos
Multimerização Proteica , Subunidades Proteicas/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Domínios Proteicos , Dobramento de Proteína , Subunidades Proteicas/química , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo
12.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013569

RESUMO

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.


Assuntos
Coagulação Sanguínea , Dissulfetos/química , Fator XIII/química , Subunidades Proteicas/química , Transtornos da Coagulação Sanguínea/sangue , Retículo Endoplasmático/metabolismo , Fator XIII/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
13.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 227-232, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950822

RESUMO

The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.


Assuntos
Proteínas de Bactérias/química , Burkholderia pseudomallei/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Subunidades Proteicas/química
14.
Nature ; 567(7749): 486-490, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894744

RESUMO

In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide from the inner to the outer membrane. The ATP-binding cassette transporter LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane. The mechanism of the LptB2FG-LptC complex (LptB2FGC) and the role of LptC in lipopolysaccharide transport are poorly understood. Here we characterize the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ATP-binding cassette transporters. Our results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Vanadatos/química , Vanadatos/metabolismo , Vanadatos/farmacologia
15.
Nature ; 567(7749): 550-553, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894747

RESUMO

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Lipopolissacarídeos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Vibrio cholerae/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Cristalografia por Raios X , Escherichia coli , Proteínas de Escherichia coli/química , Klebsiella pneumoniae , Lipopolissacarídeos/química , Proteínas de Membrana/química , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pseudomonas aeruginosa , Vibrio cholerae/citologia , Vibrio cholerae/metabolismo
16.
Nat Plants ; 5(3): 263-272, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30850820

RESUMO

Photosystem I (PSI) is a highly efficient natural light-energy converter, and has diverse light-harvesting antennas associated with its core in different photosynthetic organisms. In green algae, an extremely large light-harvesting complex I (LHCI) captures and transfers energy to the PSI core. Here, we report the structure of PSI-LHCI from a green alga Bryopsis corticulans at 3.49 Å resolution, obtained by single-particle cryo-electron microscopy, which revealed 13 core subunits including subunits characteristic of both prokaryotes and eukaryotes, and 10 light-harvesting complex a (Lhca) antennas that form a double semi-ring and an additional Lhca dimer, including a novel four-transmembrane-helix Lhca. In total, 244 chlorophylls were identified, some of which were located at key positions for the fast energy transfer. These results provide a firm structural basis for unravelling the mechanisms of light-energy harvesting, transfer and quenching in the green algal PSI-LHCI, and important clues as to how PSI-LHCI has changed during evolution.


Assuntos
Clorófitas/química , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Clorofila/química , Clorofila/metabolismo , Clorófitas/genética , Microscopia Crioeletrônica , Transferência de Energia , Evolução Molecular , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/isolamento & purificação , Complexo de Proteína do Fotossistema I/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
17.
Nature ; 568(7751): 230-234, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30894743

RESUMO

The essential but enigmatic functions of sleep1,2 must be reflected in molecular changes sensed by the brain's sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need4, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep5. Here we show that oxidative byproducts of mitochondrial electron transport6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain8,9 of Shaker's KVß subunit, Hyperkinetic10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP+-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep-three processes implicated independently in lifespan, ageing, and degenerative disease6,12-14-are thus mechanistically connected. KVß substrates8,15,16 or inhibitors that alter the ratio of bound NADPH to NADP+ (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs.


Assuntos
Drosophila melanogaster/fisiologia , Mitocôndrias/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Subunidades Proteicas/metabolismo , Sono/fisiologia , Potenciais de Ação , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Transporte de Elétrons , Metabolismo Energético , Feminino , Proteínas Luminescentes/metabolismo , NADP/metabolismo , Neurônios/metabolismo , Optogenética , Oxirredução , Estresse Oxidativo , Oxirredutases/metabolismo , Subunidades Proteicas/química , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Medicamentos Indutores do Sono , Fatores de Tempo
18.
Phys Chem Chem Phys ; 21(9): 4822-4830, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30775748

RESUMO

Cytochrome c oxidase catalyses the terminal step of cellular respiration in eukaryotes and in many prokaryotes. This enzyme reduces molecular oxygen by means of a process coupled with proton pumping. Models for proton pumping activity in cytochrome c oxidase can be divided into two groups, which are still strongly debated to date: one in which haem a is the key player, and another where this role is covered by the oxygen reduction site. Current models share the fact of requesting, more or less explicitly, an ordered sequence of events. Here, we show that all the available subunit I structures of this enzyme can be clustered in four groups. Starting from these structural observations, and considering the large corpus of available experimental data and theoretical considerations, a simple four-state (stochastic) pump model is proposed. This model implies a series of characteristics that reflect the behavior of the real enzyme in a natural way, where strictly sequential models require ad hoc assumptions (e.g. slipping mechanisms). Our results suggest that the stochastic conformational coupling could be a mechanism for energy transduction used by the protein machines.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton , Relação Estrutura-Atividade
19.
Soft Matter ; 15(9): 1999-2008, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30719518

RESUMO

Vimentin intermediate filaments constitute a distinct filament system in mesenchymal cells that is instrumental for cellular mechanics and migration. In vitro, the rod-like monomers assemble in a multi-step, salt-dependent manner into micrometer long biopolymers. To disclose the underlying mechanisms further, we employed small angle X-ray scattering on two recombinant vimentin variants, whose assembly departs at strategic points from the normal assembly route: (i) vimentin with a tyrosine to leucine change at position 117; (ii) vimentin missing the non-α-helical carboxyl-terminal domain. Y117L vimentin assembles into unit-length filaments (ULFs) only, whereas ΔT vimentin assembles into filaments containing a higher number of tetramers per cross section than normal vimentin filaments. We show that the shape and inner structure of these mutant filaments is significantly altered. ULFs assembled from Y117L vimentin contain more, less tightly bundled vimentin tetramers, and ΔT vimentin filaments preserve the number density despite the higher number of tetramers per filament cross-section.


Assuntos
Filamentos Intermediários/metabolismo , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Vimentina/química , Vimentina/genética , Humanos , Espalhamento a Baixo Ângulo , Difração de Raios X
20.
Methods Mol Biol ; 1949: 213-235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790259

RESUMO

Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Bactérias/genética , Transporte Biológico , Proteínas de Transporte/isolamento & purificação , Cromatografia Líquida , Cromatografia em Camada Delgada , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Lipossomos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Leveduras/genética
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