RESUMO
Playing a pivotal role in the metabolism of neurotransmitters in the central nervous system, the mitochondrial enzymes monoamine oxidases A and B (MAO A and B) have been for long studied as drug targets for neurodegenerative and neurological diseases. MAO inhibitors (MAOIs) are clinically used to treat Parkinson's disease and depression by blocking the degradation of neuroactive catecholamines and providing a symptomatic relief in the patients. More recent is the idea that the neuroprotective effect of MAOIs may result from the prevention of oxidative stress produced by the MAO reaction rather than being simply related to the inhibition of neurotransmitters degradation. Tranylcypromine and phenelzine are among the first developed MAOI drugs and have been used for years to treat depression. Their usage is now limited to cases of refractory depression because of their negative side effects, which are due to both the lack of MAO A/MAO B selectivity and the inhibition of other enzymes such as the drug-metabolizing cytochromes P450. Although the multi-target action of these MAOIs determines negative implications, the most newly developed compounds have improved properties not only for their specificity relatively to MAO A/MAO B selectivity but also because they function through multiple mechanisms that produce beneficial effects. In particular, safinamide, a MAO B selective inhibitor in clinical trials for Parkinson's disease, is neuroprotective by blocking the voltage-dependent Na+ and Ca2+ channels and the Ca2+-mediated glutamate release processes. Rasagiline is a drug used in combination with L-dopa in the treatment of parkinsonian patients and the metabolic products of its degradation exert neuroprotective effects. Moreover, rasagiline scaffold is used to design analogs by addition of pharmacophores that act on other neurological targets. This multi-target approach may prove successful in order to find new and more effective therapies for the complexity of neurodegenerative diseases.
Assuntos
Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Animais , Humanos , Inibidores da Monoaminoxidase/química , Doenças do Sistema Nervoso/enzimologia , Relação Estrutura-AtividadeRESUMO
Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K(i) of 8.3 ± 0.6 µM, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K(d) of 9 ± 2 nM, representing an increase in binding energy Δ(ΔG) of -3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln(206) and a "closed conformation" of the side chain of Ile(199), forming a hydrophobic "sandwich" with the side chain of Ile(316) on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile(316) is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.
Assuntos
Benzofuranos/metabolismo , Imidazóis/metabolismo , Monoaminoxidase/química , Monoaminoxidase/metabolismo , Proteínas Recombinantes/metabolismo , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Humanos , Monoaminoxidase/genética , Inibidores da Monoaminoxidase/farmacologia , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tranilcipromina/farmacologiaRESUMO
In living organisms, genes encoding proteins that contain flavins as a prosthetic group constitute approximately 2-3% of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here, we present a modified Thermofluor approach called ThermoFAD (Thermofluor-adapted flavin ad hoc detection system), which simplifies identification of optimal purification and storage conditions as well as high-affinity ligands. In this technique, the flavin cofactor is used as an intrinsic probe to monitor protein folding and stability, taking advantage of the different fluorescent properties of flavin-containing proteins between the folded and denatured state. The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and standard laboratory equipment. We have explored several cases that demonstrate the reliability and versatility of this technique when applied to globular flavoenzymes, membrane-anchored flavoproteins, and macromolecular complexes. The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis, including crystallisation. The method is likely to be applicable to other classes of proteins that possess endogenous fluorescent cofactors and prosthetic groups.
Assuntos
Flavinas/química , Dobramento de Proteína , Detergentes/química , Lignanas , Modelos Moleculares , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.
Assuntos
Mamastrovirus/enzimologia , Serina Endopeptidases/química , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Primers do DNA/genética , Humanos , Mamastrovirus/classificação , Mamastrovirus/genética , Mamastrovirus/patogenicidade , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Eletricidade EstáticaRESUMO
Mechanistic and structural studies have been carried out to investigate the molecular basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent K(i) of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 molar stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (lambda(max) approximately 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV circular dichroism spectra of the mofegiline-MAO-B adduct shows a negative peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The X-ray crystal structure of the mofegiline-MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.
Assuntos
Compostos Alílicos/química , Compostos Alílicos/farmacologia , Butilaminas/química , Butilaminas/farmacologia , Química Farmacêutica/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Monoaminoxidase/química , Proteínas Recombinantes/química , Animais , Carbono/química , Dicroísmo Circular , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Conformação Molecular , RatosAssuntos
Colletotrichum/enzimologia , Proteínas Fúngicas/química , Poligalacturonase/química , Sequência de Bases , Colletotrichum/genética , Cristalografia por Raios X , Primers do DNA , Proteínas Fúngicas/genética , Plantas/microbiologia , Poligalacturonase/antagonistas & inibidores , Poligalacturonase/isolamento & purificação , Conformação ProteicaRESUMO
GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Ochrobactrum anthropi/enzimologia , Serina/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Dimerização , Estabilidade Enzimática , Glutationa Transferase/classificação , Glutationa Transferase/genética , Cinética , Modelos Moleculares , Mutação/genética , Ochrobactrum anthropi/genética , Dobramento de Proteína , Estrutura Quaternária de Proteína , Serina/genética , Homologia Estrutural de Proteína , Especificidade por Substrato , TemperaturaRESUMO
Polygalacturonase-inhibiting proteins are plant extracellular leucine-rich repeat proteins that specifically bind and inhibit fungal polygalacturonases. The interaction with PGIP limits the destructive potential of polygalacturonases and might trigger the plant defence responses induced by oligogalacturonides. A high degree of polymorphism is found both in PGs and PGIPs, accounting for the specificity of different plant inhibitors for PGs from different fungi. Here, we review the structural features and our current understanding of the PG-PGIP interaction.
Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Animais , Humanos , Leucina/química , Modelos Moleculares , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Poligalacturonase/química , Poligalacturonase/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c(551) (Phe(7) to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three "bound" water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability.
Assuntos
Citocromos c/química , Engenharia de Proteínas , Dobramento de Proteína , Cristalografia por Raios X , Cinética , Mutagênese , Mutação Puntual , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologiaRESUMO
The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c551, which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.
Assuntos
Bactérias/química , Grupo dos Citocromos c/química , Cristalografia por Raios X , Compostos Férricos/química , Cinética , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.