RESUMO
As metalloproteins exemplify, the chemical and physical properties of metal centers depend not only on their first but also on their second coordination sphere. Installing arrays of functional groups around the first coordination sphere of synthetic metal complexes is thus highly desirable, but it remains a challenging objective. Here we introduce a novel approach to produce tailored second coordination spheres. We used bioinspired artificial architectures based on aromatic oligoamide foldamers to construct a rigid, modular and well-defined environment around a metal complex. Specifically, aza-aromatic monomers having a tethered [2Fe-2S] cluster have been synthesized and incorporated in conical helical foldamer sequences. Exploiting the modularity and predictability of aromatic oligoamide structures allowed for the straightforward design of a conical architecture able to sequester the metal complex in a confined environment. Even though no direct metal complex-foldamer interactions were purposely designed in this first generation model, crystallography, NMR and IR spectroscopy concurred to show that the aromatic oligoamide backbone alters the structure and fluxional processes of the metal cluster.
RESUMO
The development of large synthetic ligands could be useful to target the sizeable surface areas involved in protein-protein interactions. Herein, we present long helical aromatic oligoamide foldamers bearing proteinogenic side chains that cover up to 450â Å2 of the human carbonic anhydraseâ II (HCA) surface. The foldamers are composed of aminoquinolinecarboxylic acids bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids that enhance helix handedness dynamics. Crystal structures of HCA-foldamer complexes were obtained with a 9- and a 14-mer both showing extensive protein-foldamer hydrophobic contacts. In addition, foldamer-foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Solution studies confirm the positioning of the foldamer at the protein surface as well as a dimerization of the complexes.
RESUMO
Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.
RESUMO
Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.
Assuntos
Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Simulação de Acoplamento Molecular , Domínios ProteicosRESUMO
The promotion of protein dimerization using the aggregation properties of a protein ligand was explored and shown to produce complexes with unusual stoichiometries. Helical foldamer 2 was synthesized and bound to human carbonic anhydrase (HCA) using a nanomolar active site ligand. Crystal structures show that the hydrophobicity of 2 and interactions of its side chains lead to the formation of an HCA2-23 complex in which three helices of 2 are stacked, two of them being linked to an HCA molecule. The middle foldamer in the stack can be replaced by alternate sequences 3 or 5. Solution studies by CD and NMR confirm left-handedness of the helical foldamers as well as HCA dimerization.
Assuntos
Anidrases Carbônicas/química , Hidrocarbonetos Aromáticos/química , Anidrases Carbônicas/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Estrutura MolecularRESUMO
Hydroperoxides can act as specific haptens and oxidatively modify proteins. Terpene hydroperoxides trigger unusually high frequencies of positive skin reactions in human patients if tested at high concentrations. It is unknown whether this is due to specific hapten formation. Here, we show that both terpene hydroperoxides and the endogenous hydroperoxide formed from squalene can oxidatively modify tryptophan. Oxidative modifications of Trp were recently postulated to explain cross-sensitization between unrelated photosensitizers. Current observations may extend this hypothesis: Oxidative events triggered by endogenous hydroperoxides and hydroperoxides/oxidants derived from xenobiotics might lead to a sensitized state detected by patch tests with high concentrations of hydroperoxides.
Assuntos
Testes Diagnósticos de Rotina , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Triptofano/metabolismo , Estrutura Molecular , Oxirredução/efeitos dos fármacosRESUMO
A rational approach for the construction of multi-stranded artificial ß-sheets based not on hydrogen bonding, but rather on π-π aromatic stacking, is presented. Using 4,6-dinitro-1,3-phenylenediamine units, rigid turns were designed that allow face-to-face π-π interactions between appended linear aromatic segments to be strong enough for folding in an organic solvent, but weak enough to prevent aggregation and precipitation. Solution and solid-state studies on a series of turn units showed that the desired degree of rigidity, resulting from hindered bond rotation, could be fine-tuned by the inclusion of additional methyl substituents on the aromatic rings. The high degree of preorganization afforded by these qualities further allowed the facile preparation of macrocyclic sheet structures from their noncyclic precursors. These macrocycles were shown to have slow internal dynamics and defined conformational preferences. Using this background, three- and five-stranded artificial ß-sheets were synthesized and their folded conformations extensively characterized in solution by NMR. The solid-state structures of the three- and five-stranded sheets were also elucidated in the solid state by X-ray crystallography and confirmed intramolecular π-π aromatic stacking.
Assuntos
Amidas/química , Hidrocarbonetos Aromáticos/química , Compostos Macrocíclicos/química , Cristalografia por Raios X , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação MolecularRESUMO
In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions.
Assuntos
Amidas/química , Anidrase Carbônica II/química , Amidas/metabolismo , Sítios de Ligação , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. RESULTS: In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. CONCLUSIONS: Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.
Assuntos
Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Vitis/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flavonoides/biossíntese , Flavonoides/genética , Regulação da Expressão Gênica de Plantas , Genes myb , Modelos Moleculares , Dados de Sequência Molecular , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estrutura Terciária de Proteína , RNA de Plantas/genética , Nicotiana/genética , Nicotiana/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Vitis/metabolismoRESUMO
Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca(2+) activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
Assuntos
Anexina A5/química , Anexina A5/metabolismo , Membrana Celular/fisiologia , Cicatrização , Animais , Anexina A5/genética , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/genética , Camundongos , Camundongos KnockoutRESUMO
(E)-4-(ethoxymethylene)-2-phenyloxazol-5(4H)-one, commonly referred to as oxazolone, is the most potent skin sensitizer in published databases as determined with the murine local lymph node assay. It has been used very widely in immunological research to induce and elicit skin sensitization reactions in experimental animals. Nevertheless, no detailed study on the reactivity of oxazolone with proteins or peptides has been published, which would rationalize its unique sensitization potential from a chemical point of view. Peptide reactivity assays have been proposed as alternatives to animal tests to study the skin sensitization potential of test chemicals. Besides their application to reduce animal experimentation, peptide reactivity assays also allow one to gain mechanistic insights into the reactivity of test chemicals with proteins. In this case study, we applied different peptide reactivity assays to investigate and mechanistically rationalize the reactivity of oxazolone. Its sensitization potential could be linked to the following findings: (i) oxazolone reacts rapidly with the amine group in lysine with an addition-elimination reaction at the ethoxymethylene group to form stable products within minutes at physiological pH; (ii) sequentially different products with cysteine-peptides are formed, the most stable being an S-hippuryl-modification; and (iii) the S-hippuryl-modification can be shuttled to other nucleophilic sites; thus, also Lys residues can subsequently be modified with a hippuryl-moiety. This very rapid and diverse reactivity especially with lysine residues may explain why oxazolone forms sufficient stable novel epitopes on proteins to induce skin sensitization even at very low concentration.
Assuntos
Oxazolona/análogos & derivados , Oxazolona/química , Oxazolona/farmacologia , Peptídeos/química , Pele/efeitos dos fármacos , Animais , Cisteína/química , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Irritantes/síntese química , Irritantes/química , Irritantes/farmacologia , Cinética , Lisina/análogos & derivados , Lisina/química , Camundongos , Oxazolona/síntese química , Compostos de Sulfidrila/químicaRESUMO
Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica/métodos , Proteínas de Membrana Transportadoras/ultraestrutura , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Resistência a Múltiplos Medicamentos , Tomografia com Microscopia Eletrônica/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Pseudomonas aeruginosa/metabolismoRESUMO
Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 A. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 A, indicates the ordering of a short 3(10) helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser...TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.
Assuntos
Antocianinas/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Vitis/enzimologia , Sítios de Ligação , Coenzimas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , NADP/metabolismo , Oxirredução , Ligação ProteicaRESUMO
Nucleotide insertions that modify the C terminus of ferritin light chain (FTL) cause neurodegenerative movement disorders named neuroferritinopathies, which are inherited with dominant transmission. The disorders are characterized by abnormal brain iron accumulation. Here we describe the biochemical and crystallographic characterization of pathogenic FTL mutant p.Phe167SerfsX26 showing that it is a functional ferritin with an altered conformation of the C terminus. Moreover we analyze functional and stability properties of ferritin heteropolymers made of 20-23 H-chains and 1-4 L-chains with representative pathogenic mutations or the last 10-28 residues truncated. All the heteropolymers containing the pathogenic or truncated mutants had a strongly reduced capacity to incorporate iron, both when expressed in Escherichia coli, and in vitro when iron was supplied as Fe(III) in the presence of ascorbate. The mutations also reduced the physical stability of the heteropolymers. The data indicate that even a few mutated L-chains are sufficient to alter the permeability of 1-2 of the 6 hydrophobic channels and modify ferritin capacity to incorporate iron. The dominant-negative action of the mutations explains the dominant transmission of the disorder. The data support the hypothesis that hereditary ferritinopathies are due to alterations of ferritin functionality and provide new input on the mechanism of the function of isoferritins.
Assuntos
Apoferritinas/genética , Apoferritinas/metabolismo , Ferro/metabolismo , Mutação , Degeneração Neural/genética , Degeneração Neural/metabolismo , Sequência de Aminoácidos , Apoferritinas/química , Cristalografia por Raios X , Genes Dominantes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Degeneração Neural/etiologia , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletricidade EstáticaRESUMO
Together with leucoanthocyanidin reductase, anthocyanidin reductase (ANR) is one of the two enzymes of the flavonoid-biosynthesis pathway that produces the flavan-3-ol monomers required for the formation of proanthocyanidins or condensed tannins. It has been shown to catalyse the double reduction of anthocyanidins to form 2R,3R-flavan-3-ols, which can be further transformed to the 2S,3R isomers by non-enzymatic epimerization. ANR from grape (Vitis vinifera) was expressed in Escherichia coli and purified. Unexpectedly, RP-HPLC, LC-MS and NMR experiments clearly established that the enzyme produces a 50:50 mixture of 2,3-cis and 2,3-trans flavan-3-ols which have been identified by chiral chromatography to be 2S,3S- and 2S,3R-flavan-3-ols, i.e. the naturally rare (+)-epicatechin and (-)-catechin, when cyanidin is used as the substrate of the reaction. The first three-dimensional structure of ANR is described at a resolution of 2.2 A and explains the inactivity of the enzyme in the presence of high salt concentrations.
Assuntos
Regulação Alostérica , Antocianinas/metabolismo , NADH NADPH Oxirredutases/química , Racemases e Epimerases/química , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Isomerismo , NADH NADPH Oxirredutases/genética , Oxirredução , Conformação Proteica , Racemases e Epimerases/genética , Relação Estrutura-Atividade , Transgenes/genética , Vitis/enzimologiaRESUMO
Annexins are soluble proteins that bind to biological membranes in a Ca(2+)-dependent manner. Annexin-A6 (AnxA6) is unique in the annexin family as it consists of the repeat of two annexin core modules, while all other annexins consist of a single module. AnxA6 has been proposed to participate in various membrane-related processes, including endocytosis and exocytosis, yet the molecular mechanism of association of AnxA6 with biological membranes, especially its ability to aggregate membranes, is still unclear. To address this question, we studied the association of AnxA6 with model phospholipid membranes by combining the techniques of quartz crystal microbalance with dissipation monitoring (QCM-D), (cryo-) transmission electron microscopy (TEM) and atomic force microscopy (AFM). The properties of membrane binding and membrane aggregation of AnxA6 were compared to two reference systems, annexin A5 (AnxA5), which is the annexin prototype, and a chimerical AnxA5-dimer molecule, which is able to aggregate two membranes in a symmetrical manner. We show that AnxA6 presents two modes of association with lipid membranes depending on Ca(2+)-concentration. At low Ca(2+)-concentration ( approximately 60-150microM), AnxA6 binds to membranes via its two coplanar annexin modules and is not able to associate two separate membranes. At high Ca(2+)-concentration ( approximately 2mM), AnxA6 molecules are able to bind two adjacent phospholipid membranes and present a conformation similar to the AnxA6 3D crystallographic structure. Possible biological implications of these novel membrane-binding properties of AnxA6 are discussed.
Assuntos
Anexina A6/metabolismo , Microscopia Crioeletrônica/métodos , Membranas Artificiais , Microscopia de Força Atômica/métodos , Fosfolipídeos/química , Proteínas Recombinantes/metabolismo , Anexina A5/genética , Anexina A5/metabolismo , Anexina A5/ultraestrutura , Anexina A6/genética , Anexina A6/ultraestrutura , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestruturaRESUMO
Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid biosynthesis pathway which catalyses the NADPH-dependent reduction of 2R,3R-trans-dihydroflavonols to leucoanthocyanidins. The latter are the precursors of anthocyans and condensed tannins, two major classes of phenolic compounds that strongly influence the organoleptic properties of wine. DFR has been investigated in many plant species, but little was known about its structural properties until the three-dimensional structure of the Vitis vinifera enzyme complexed with NADP(+) and its natural substrate dihydroquercetin (DHQ) was described. In the course of the study of substrate specificity, crystals of DFR-NADP(+)-flavonol (myricetin and quercetin) complexes were obtained. Their structures exhibit major changes with respect to that of the abortive DFR-NADP(+)-DHQ complex. Two flavonol molecules bind to the catalytic site in a stacking arrangement and alter its geometry, which becomes incompatible with enzymatic activity. The X-ray structures of both DFR-NADP(+)-myricetin and DFR-NADP(+)-quercetin are reported together with preliminary spectroscopic data. The results suggest that flavonols could be inhibitors of the activity of DFR towards dihydroflavonols.
Assuntos
Oxirredutases do Álcool/química , Flavonoides/química , Proteínas de Plantas/química , Quercetina/química , Vitis/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Flavonoides/biossíntese , Modelos Moleculares , NADP/químicaRESUMO
The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 A resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.
Assuntos
Oxirredutases do Álcool/química , Flavonoides/biossíntese , Proteínas de Plantas/química , Estrutura Terciária de Proteína , Vitis/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , NADP/química , NADP/metabolismo , Oxirredução , Proteínas de Plantas/metabolismoRESUMO
Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38 Angstrom resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (approximately 80%). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced ( approximately 41%) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.