RESUMO
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.
Assuntos
Corynebacterium pseudotuberculosis/metabolismo , Vitamina B 12/metabolismo , Adenina/química , Adenina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinética , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometria de Fluorescência , Homologia Estrutural de Proteína , Suramina/química , Suramina/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/químicaRESUMO
In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens. The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.
Assuntos
Corynebacterium pseudotuberculosis/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Polímeros/química , Polímeros/farmacologia , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/química , Domínio Catalítico , Corynebacterium pseudotuberculosis/genética , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxirredução , Polieletrólitos , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificaçãoRESUMO
Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.
Assuntos
Exfoliatinas/química , Exfoliatinas/toxicidade , Staphylococcus aureus/química , Staphylococcus aureus/patogenicidade , Animais , Cristalografia por Raios X , Exfoliatinas/classificação , Feminino , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Ovinos , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/microbiologia , Eletricidade Estática , Homologia Estrutural de Proteína , SíndromeRESUMO
The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable economical loss. Cold shock proteins are conserved in many bacteria and eukaryotic cells and they help to restore normal cell functions after cold shock in which some appear to have specific functions at normal growth temperature as well. Cold shock protein A from C. pseudotuberculosis was expressed in Escherichia coli and purified. The thermal unfolding/refolding process characterized by circular dichroism, differential scanning calorimetry and NMR spectroscopy techniques indicated that the refolding process was almost completely reversible.
Assuntos
Proteínas e Peptídeos de Choque Frio/química , Proteínas e Peptídeos de Choque Frio/genética , Corynebacterium pseudotuberculosis/genética , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Clonagem Molecular , Proteínas e Peptídeos de Choque Frio/isolamento & purificação , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/veterinária , Escherichia coli/genética , Cabras , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Redobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Ovinos , Temperatura de TransiçãoRESUMO
Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now integrated into the BlueStar STING suite of programs. Consequently, the prediction of protein-protein interfaces for all proteins available in the PDB is possible through STING_interfaces module, accessible at the following website: (http://www.cbi.cnptia.embrapa.br/SMS/predictions/index.html).