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1.
Proc Natl Acad Sci U S A ; 116(51): 25649-25658, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31757855

RESUMO

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modeling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multiscale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregates in the stalks formed between 2 opposing lipid bilayers. Infection of macrophages pretreated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological response of macrophages.


Assuntos
Lipídeos/química , Macrófagos/microbiologia , Mycobacterium tuberculosis , Tuberculose/microbiologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Macrófagos/química , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , Ressonância Magnética Nuclear Biomolecular
2.
Proc Natl Acad Sci U S A ; 116(35): 17525-17530, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31416915

RESUMO

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.


Assuntos
Grelina/química , Grelina/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Acilação , Animais , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
3.
Bioorg Chem ; 114: 105021, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34120023

RESUMO

The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.


Assuntos
Antineoplásicos/química , Bibliotecas de Moléculas Pequenas/química , Fator de Transcrição TFIIH/antagonistas & inibidores , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidade , Eletricidade Estática , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo
4.
Proc Natl Acad Sci U S A ; 114(16): 4231-4236, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28373551

RESUMO

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Corynebacterium glutamicum/metabolismo , Bicamadas Lipídicas/metabolismo , Ácidos Micólicos/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Corynebacterium glutamicum/crescimento & desenvolvimento , Transporte Proteico , Homologia de Sequência
5.
J Biol Chem ; 293(39): 14974-14988, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30068551

RESUMO

The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas de Neoplasias/química , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Fator de Transcrição TFIIH/química , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cristalografia por Raios X , Reparo do DNA/efeitos dos fármacos , Dimerização , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição TFIIH/genética
6.
Proc Natl Acad Sci U S A ; 112(38): 11852-7, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26372966

RESUMO

The structure of the dynorphin (1-13) peptide (dynorphin) bound to the human kappa opioid receptor (KOR) has been determined by liquid-state NMR spectroscopy. (1)H and (15)N chemical shift variations indicated that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM KOR. Radioligand binding indicated an intermediate-affinity interaction, with a Kd of ∼200 nM. Transferred nuclear Overhauser enhancement spectroscopy was used to determine the structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly disordered, the central part of the peptide from L5 to R9 to form a helical turn, and the C-terminal segment from P10 to K13 to be flexibly disordered in this intermediate-affinity bound state. Combining molecular modeling with NMR provided an initial framework for understanding multistep activation of a G protein-coupled receptor by its cognate peptide ligand.


Assuntos
Dinorfinas/química , Dinorfinas/metabolismo , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Sequência de Aminoácidos , Dinorfinas/isolamento & purificação , Humanos , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Isótopos de Nitrogênio , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos , Piperidinas/química , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Opioides kappa/química , Tetra-Hidroisoquinolinas/química , Fatores de Tempo
7.
J Biol Chem ; 291(17): 9042-51, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26895963

RESUMO

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a (13)C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Methylobacterium extorquens/química , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Methylobacterium extorquens/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
8.
J Am Chem Soc ; 139(4): 1590-1597, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28059506

RESUMO

The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the ß-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Klebsiella pneumoniae/química , Bicamadas Lipídicas/química , Termodinâmica , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Klebsiella pneumoniae/metabolismo , Bicamadas Lipídicas/metabolismo , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
J Struct Biol ; 194(3): 337-46, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26975212

RESUMO

Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features.


Assuntos
Multimerização Proteica , Proteínas Repressoras/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos/fisiologia , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Repressoras/fisiologia
10.
Biochim Biophys Acta ; 1840(1): 626-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24140392

RESUMO

BACKGROUND: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition. METHODS: Detailed kinetic analysis (kcat, KM, pKa determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution. RESULTS: The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pKa of the catalytic nucleophile (E298), the pKa of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants. CONCLUSIONS AND GENERAL SIGNIFICANCE: Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 α-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.


Assuntos
Arabinose/metabolismo , Bacillaceae/enzimologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mutação/genética , Bacillaceae/genética , Bacillaceae/metabolismo , Catálise , Domínio Catalítico , Cromatografia em Camada Fina , Glicosídeo Hidrolases/química , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade por Substrato
11.
Biophys J ; 107(10): 2305-12, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25418299

RESUMO

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (ß2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in ß2AR. By analyzing the ß2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and ß2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.


Assuntos
Colesterol/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Especificidade por Substrato , Temperatura
12.
Biochim Biophys Acta ; 1828(9): 2173-81, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23643889

RESUMO

Cord factor (trehalose 6,6'-dimycolate, TDM) is the major lipid in the outer membrane of Corynebacteria and Mycobacteria. Although its role is well recognized in the immune response phenomena, its membrane biophysical properties remained largely unexplored and TDM has often been described as a detergent. We purified the main components of the outer membrane from Corynebacterium glutamicum and analyzed their membrane forming properties. In mixture with endogenous cardiolipin, but not alone, the spontaneous hydration of TDM produces liposomes. As a pure component, TDM formed vesicles only by the detergent dialysis method. Perdeuterated cardiolipin-TDM mixtures were shown by deuterium nuclear magnetic resonance (NMR) to exhibit a gel to liquid crystalline phase transition over a 273-295K temperature range, for cells grown at 303K, and thus to be in a liquid crystalline state at physiological temperature. Molecular dynamics simulations of hydrated TDM bilayers provided the trehalose average orientation and conformation, the chain order parameters, the area per lipid and the bilayer thickness which was confirmed by electron microscopy. Finally the Porin A-Porin H ion channel from the Corynebacterial outer membrane was reconstituted in TDM liposomes. With properly mycoloylated proteins, it manifested the typical voltage dependent ion channel properties of an outer membrane porin.


Assuntos
Membrana Celular/química , Fatores Corda/química , Bicamadas Lipídicas/química , Lipossomos/química , Porinas/química , Cardiolipinas/química , Membrana Celular/ultraestrutura , Fatores Corda/isolamento & purificação , Corynebacterium glutamicum/química , Deutério , Canais Iônicos/química , Lipossomos/ultraestrutura , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Transição de Fase , Porinas/isolamento & purificação , Temperatura
13.
J Membr Biol ; 247(9-10): 827-42, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24676477

RESUMO

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Polímeros/química , Tensoativos/química , Animais , Artefatos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Solubilidade , Soluções , Avaliação da Tecnologia Biomédica , Água/química
14.
Nucleic Acids Res ; 40(19): 9927-40, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22844099

RESUMO

The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Distúrbios Distônicos/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Termodinâmica
15.
Orig Life Evol Biosph ; 44(3): 197-208, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25351682

RESUMO

Terpenoids have an essential function in present-day cellular membranes, either as membrane reinforcers in Eucarya and Bacteria or as principal membrane constituents in Archaea. We have shown that some terpenoids, such as cholesterol and α, ω-dipolar carotenoids reinforce lipid membranes by measuring the water permeability of unilamellar vesicles. It was possible to arrange the known membrane terpenoids in a 'phylogenetic' sequence, and a retrograde analysis led us to conceive that single-chain polyprenyl phosphates might have been 'primitive' membrane constituents. By using an optical microscopy, we have observed that polyprenyl phosphates containing 15 to 30 C-atoms form giant vesicles in water in a wide pH range. The addition of 10 % molar of some polyprenols to polyprenyl phosphate vesicles have been shown to reduce the water permeability of membranes even more efficiently than the equimolecular addition of cholesterol. A 'prebiotic' synthesis of C10 and C15 prenols from C5 monoprenols was achieved in the presence of a montmorillonite clay. Hypothetical pathway from C1 or C2 units to 'primitive' membranes and that from 'primitive' membranes to archaeal lipids are presented.


Assuntos
Archaea/química , Membrana Celular/química , Evolução Molecular , Fosfatos de Poli-Isoprenil/química , Silicatos de Alumínio , Bactérias/química , Bentonita , Carotenoides/química , Permeabilidade da Membrana Celular , Colesterol/química , Argila , Eucariotos/química , Concentração de Íons de Hidrogênio , Origem da Vida , Terpenos/química , Lipossomas Unilamelares/química , Água/química
16.
Biochim Biophys Acta ; 1818(9): 2344-53, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22579977

RESUMO

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded ß-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the ß-barrel and loop regions were probed by the spin-lattice relaxation times of the C(α) and C(ß) atoms of the serine and threonine residues, and by cross-polarization dynamics. The ß-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/fisiologia , Klebsiella pneumoniae/metabolismo , Sequência de Aminoácidos , Centrifugação com Gradiente de Concentração , Detergentes/química , Escherichia coli/metabolismo , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Micelas , Microscopia Eletrônica de Transmissão/métodos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sacarose/química
17.
J Biomol NMR ; 56(1): 3-15, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23306615

RESUMO

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ligação a DNA/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Homologia de Sequência de Aminoácidos
18.
Langmuir ; 29(25): 8031-8, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23763339

RESUMO

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.


Assuntos
Colesterol/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos
19.
Cell Rep ; 42(4): 112320, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37027306

RESUMO

The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.


Assuntos
Receptores de Grelina , Sódio , Regulação Alostérica , Sítio Alostérico , Grelina/metabolismo , Íons , Receptores de Grelina/metabolismo , Transdução de Sinais , Sódio/metabolismo
20.
J Biol Chem ; 286(37): 32525-32, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21799011

RESUMO

PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. (15)N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.


Assuntos
Proteínas de Bactérias/biossíntese , Corynebacterium glutamicum , Expressão Gênica , Proteínas de Membrana/biossíntese , Ácidos Micólicos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Bactérias/genética , Escherichia coli , Proteínas de Membrana/genética , Ressonância Magnética Nuclear Biomolecular
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