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1.
J Biol Inorg Chem ; 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31853648

RESUMO

Nickel ions are crucial components for the catalysis of biological reactions in prokaryotic organisms. As an uncontrolled nickel trafficking is toxic for living organisms, nickel-dependent bacteria have developed tightly regulated strategies to maintain the correct intracellular metal ion quota. These mechanisms require transcriptional regulator proteins that respond to nickel concentration, activating or repressing the expression of specific proteins related to Ni(II) metabolism. In Streptomyces griseus, a Gram-positive bacterium used for antibiotic production, SgSrnR and SgSrnQ regulate the nickel-dependent antagonistic expression of two superoxide dismutase (SOD) enzymes, a Ni-SOD and a FeZn-SOD. According to a previously proposed model, SgSrnR and SgSrnQ form a protein complex in which SgSrnR works as repressor, binding directly to the promoter of the gene coding for FeZn-SOD, while SgSrnQ is the Ni(II)-dependent co-repressor. The present work focuses on the determination of the biophysical and functional properties of SgSrnR. The protein was heterologously expressed and purified from Escherichia coli. The structural and metal-binding analysis, carried out by circular dichroism, light scattering, fluorescence and isothermal titration calorimetry, showed that the protein is a well-structured homodimer, able to bind nickel with moderate affinity. DNase I footprinting and ß-galactosidase gene reporter assays revealed that apo-SgSrnR is able to bind its DNA operator and activates a transcriptional response. The structural and functional properties of this protein are discussed relatively to its role as a Ni(II)-dependent sensor.

2.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671552

RESUMO

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

3.
Chemistry ; 25(67): 15351-15360, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31486181

RESUMO

In Rhodospirillum rubrum, the maturation of carbon monoxide dehydrogenase (CODH) requires three nickel chaperones, namely RrCooC, RrCooT and RrCooJ. Recently, the biophysical characterisation of the RrCooT homodimer and the X-ray structure of its apo form revealed the existence of a solvent-exposed NiII -binding site at the dimer interface, involving the strictly conserved Cys2. Here, a multifaceted approach that used NMR and X-ray absorption spectroscopies, complemented with structural bio-modelling methodologies, was used to characterise the binding mode of NiII in RrCooT. This study suggests that NiII adopts a square-planar geometry through a N2 S2 coordinating environment that comprises the two thiolate and amidate groups of both Cys2 residues at the dimer interface. The existence of a diamagnetic mononuclear NiII centre with bis-amidate/bis-thiolate ligands, coordinated by a single-cysteine motif, is unprecedented in biology and raises the question of its role in the activation of CODH at the molecular level.


Assuntos
Cisteína/química , Metalochaperonas/química , Níquel/química , Rhodospirillum rubrum/química , Sequência de Aminoácidos , Sítios de Ligação , Cátions Bivalentes/química , Complexos de Coordenação/química , Ligantes , Modelos Moleculares , Nitrogênio/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Solventes/química , Enxofre/química , Termodinâmica
4.
Biochim Biophys Acta Gen Subj ; 1863(6): 1040-1049, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30928490

RESUMO

BACKGROUND: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. METHODS: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. RESULTS: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. CONCLUSIONS: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. GENERAL SIGNIFICANCE: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.


Assuntos
Acetilcoenzima A/química , Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina/metabolismo , Cinética , Ligação Proteica
5.
Methods Mol Biol ; 1964: 251-266, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929248

RESUMO

Life is governed by a complex and tightly regulated sequence of biochemical reactions, catalyzed by enzymes. Characterizing enzyme activity is extremely important both to understand biological processes and to develop new industrial applications. Calorimetry represents an ideal system to measure kinetics of biochemical transformations, because it uses heat, always produced or absorbed during chemical reactions, as a probe.The following protocol describes the details of experimental setup and data analysis of isothermal titration calorimetry (ITC) experiments aimed to quantify the thermodynamic (ΔH) and kinetic (KM and kcat) parameters of enzyme catalysis. A general guideline to choose the right procedure according to the system under analysis is given, together with some instructions on how to adjust the experimental conditions for obtaining reliable data. The method to analyze the obtained raw ITC curves and to derive the kinetic parameters is described.


Assuntos
Calorimetria/métodos , Enzimas/química , Termodinâmica , Catálise , Cinética
6.
J Biol Chem ; 294(19): 7601-7614, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30858174

RESUMO

Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallochaperones involved in nickel delivery. The maturation of carbon monoxide dehydrogenase in the proteobacterium Rhodospirillum rubrum requires three accessory proteins, CooC, CooT, and CooJ, dedicated to nickel insertion into the active site, a distorted [NiFe3S4] cluster coordinated to an iron site. Previously, CooJ from R. rubrum (RrCooJ) has been described as a nickel chaperone with 16 histidines and 2 cysteines at its C terminus. Here, the X-ray structure of a truncated version of RrCooJ, combined with small-angle X-ray scattering data and a modeling study of the full-length protein, revealed a homodimer comprising a coiled coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer) involving the His-rich motifs and having similar metal affinity (KD = 1.6 µm). Remarkably, biophysical approaches, site-directed mutagenesis, and X-ray crystallography uncovered an additional nickel-binding site at the dimer interface, which binds Ni(II) with an affinity of 380 nm Although RrCooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved H(W/F)X 2HX 3H motif, identified in this study, suggesting a dual function for CooJ both as a nickel chaperone and as a nickel storage protein.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Níquel/química , Multimerização Proteica , Rhodospirillum rubrum/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/genética , Mutagênese Sítio-Dirigida , Rhodospirillum rubrum/genética
7.
J Biol Inorg Chem ; 23(8): 1309-1330, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30264175

RESUMO

Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3-7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.


Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/química , Metalochaperonas/metabolismo , Níquel/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Teoria da Densidade Funcional , Escherichia coli/genética , Glicina/genética , Concentração de Íons de Hidrogênio , Metalochaperonas/química , Metalochaperonas/genética , Modelos Químicos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Níquel/química , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Zinco/química , Zinco/metabolismo
8.
Front Mol Neurosci ; 11: 280, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30177872

RESUMO

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.

9.
Sci Rep ; 8(1): 13716, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209332

RESUMO

Glutaredoxins (Grx) are small proteins conserved throughout all the kingdoms of life that are engaged in a wide variety of biological processes and share a common thioredoxin-fold. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (FeS) metabolism. They contain a single thiol group in their active site and use low molecular mass thiols such as glutathione as ligand for binding FeS-clusters. In this study, we investigated molecular aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei brucei, a mitochondrial class II Grx that fulfills an indispensable role in vivo. Mitochondrial 1CGrx1 from trypanosomes differs from orthologues in several features including the presence of a parasite-specific N-terminal extension (NTE) whose role has yet to be elucidated. Previously we have solved the structure of a truncated form of 1CGrx1 containing only the conserved glutaredoxin domain but lacking the NTE. Our aim here is to investigate the effect of the NTE on the conformation of the protein. We therefore solved the NMR structure of the full-length protein, which reveals subtle but significant differences with the structure of the NTE-less form. By means of different experimental approaches, the NTE proved to be intrinsically disordered and not involved in the non-redox dependent protein dimerization, as previously suggested. Interestingly, the portion comprising residues 65-76 of the NTE modulates the conformational dynamics of the glutathione-binding pocket, which may play a role in iron-sulfur cluster assembly and delivery. Furthermore, we disclosed that the class II-strictly conserved loop that precedes the active site is critical for stabilizing the protein structure. So far, this represents the first communication of a Grx containing an intrinsically disordered region that defines a new protein subgroup within class II Grx.


Assuntos
Proteínas com Ferro-Enxofre/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , Enxofre/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/fisiologia , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Oxirredução , Conformação Proteica , Multimerização Proteica/fisiologia
10.
Biochim Biophys Acta Gen Subj ; 1862(10): 2245-2253, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30048738

RESUMO

BACKGROUND: Helicobacter pylori is a bacterium strongly associated with gastric cancer. It thrives in the acidic environment of the gastric niche of large portions of the human population using a unique adaptive mechanism that involves the catalytic activity of the nickel-dependent enzyme urease. Targeting urease represents a key strategy for drug design and H. pylori eradication. METHOD: Here, we describe a novel method to screen, directly in the cellular environment, urease inhibitors. A ureolytic Escherichia coli strain was engineered by cloning the entire urease operon in an expression plasmid and used to test in-cell urease inhibition with a high-throughput colorimetric assay. A two-plasmid system was further developed to evaluate the ability of small peptides to block the protein interactions that lead to urease maturation. RESULTS: The developed assay is a robust cellular model to test, directly in the cell environment, urease inhibitors. The efficacy of a co-expressed peptide to affect the interaction between UreF and UreD, two accessory proteins necessary for urease activation, was observed. This event involves a process that occurs through folding upon binding, pointing to the importance of intrinsically disordered hot spots in protein interfaces. CONCLUSIONS: The developed system allows the concomitant screening of a large number of drug candidates that interfere with the urease activity both at the level of the enzyme catalysis and maturation. GENERAL SIGNIFICANCE: As inhibition of urease has the potential of being a global antibacterial strategy for a large number of infections, this work paves the way for the development of new candidates for antibacterial drugs.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/enzimologia , Ensaios de Triagem em Larga Escala/métodos , Urease/antagonistas & inibidores , Urease/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Inibidores Enzimáticos/química , Helicobacter pylori/genética , Níquel/metabolismo , Fragmentos de Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Urease/genética
11.
Colloids Surf B Biointerfaces ; 159: 849-860, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28892869

RESUMO

Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Micelas , Peptídeos/química , Animais , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Lipossomas Unilamelares/química
12.
Sci Rep ; 7(1): 5977, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729736

RESUMO

A growing body of literature on intrinsically disordered proteins (IDPs) led scientists to rethink the structure-function paradigm of protein folding. Enzymes are often considered an exception to the rule of intrinsic disorder (ID), believed to require a unique structure for catalysis. However, recent studies revealed the presence of disorder in several functional native enzymes. In the present work, we address the importance of dynamics for catalysis, by investigating the relationship between folding and activity in Sporosarcina pasteurii UreG (SpUreG), a P-loop GTPase and the first discovered native ID enzyme, involved in the maturation of the nickel-containing urease. The effect of denaturants and osmolytes on protein structure and activity was analyzed using circular dichroism (CD), Site-Directed Spin Labeling (SDSL) coupled to EPR spectroscopy, and enzymatic assays. Our data show that SpUreG needs a "flexibility window" to be catalytically competent, with both too low and too high mobility being detrimental for its activity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Dobramento de Proteína , Espectroscopia de Ressonância de Spin Eletrônica , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Proteínas de Ligação a Fosfato , Conformação Proteica , Desnaturação Proteica , Marcadores de Spin , Sporosarcina/enzimologia , Temperatura Ambiente
13.
Anal Bioanal Chem ; 408(28): 7971-7980, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27580605

RESUMO

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ureA ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR-OP ureA interaction. An active surface was prepared by affinity capture of OP ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR-OP ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein-dsDNA interaction. Graphical Abstract Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP ureA urease promoter immobilized on the sensor chip surface.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Helicobacter pylori/metabolismo , Níquel/metabolismo , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , DNA Bacteriano/genética , Descoberta de Drogas , Modelos Biológicos , Ligação Proteica , Proteínas Repressoras/genética , Ressonância de Plasmônio de Superfície , Titulometria , Urease/genética , Urease/metabolismo
14.
Biochim Biophys Acta ; 1864(12): 1714-1731, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27645710

RESUMO

The interplay of the presence of nickel and protein disorder in processes affecting human health is the focus of the present review. Many systems involving nickel as either a cofactor or as a toxic contaminant are characterized by large disorder. The role of nickel in the biochemistry of bacterial enzymes is discussed here, covering both the beneficial effects of nickel in the human microbiota as well as the role of nickel-depending bacteria in human pathogenesis. In addition, the hazardous health effects caused by nickel exposure to humans, namely nickel-induced carcinogenesis and allergy, are triggered by non-specific interactions of nickel with macromolecules and formation of reactive compounds that mediate cellular damage. Cellular response to nickel is also related to signal transduction cascades. This review thus highlights the most promising systems for future studies aimed at decreasing the adverse effects of nickel on human health.


Assuntos
Níquel/metabolismo , Níquel/toxicidade , Biocatálise , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Humanos , Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Níquel/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Nat Commun ; 7: 12593, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27558202

RESUMO

Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Helicobacter pylori/metabolismo , Ferro/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Íons , Substâncias Macromoleculares/metabolismo , Microscopia de Força Atômica , Modelos Biológicos , Nucleoproteínas/metabolismo , Regiões Operadoras Genéticas/genética , Ligação Proteica , Transcrição Genética/efeitos dos fármacos
16.
Methods Enzymol ; 567: 215-36, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26794356

RESUMO

Isothermal titration calorimetry (ITC) is a technique that measures the heat released or absorbed during a chemical reaction as an intrinsic probe to characterize any chemical process that involves heat changes spontaneously occurring during the reaction. The general features of this method to determine the kinetic and thermodynamic parameters of enzymatic reactions (kcat, KM, ΔH) are described and discussed here together with some detailed applications to specific cases. ITC does not require any modification or labeling of the system under analysis, can be performed in solution, and needs only small amounts of enzyme. These properties make ITC an invaluable, powerful, and unique tool to extend the knowledge of enzyme kinetics to drug discovery.


Assuntos
Calorimetria/métodos , Catálise , Cinética , L-Lactato Desidrogenase/metabolismo , Urease/metabolismo
17.
Plant Mol Biol ; 89(1-2): 49-65, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26245354

RESUMO

Intrinsically disordered proteins (IDPs) are proteins that lack secondary and/or tertiary structure under physiological conditions. These proteins are very abundant in eukaryotic proteomes and play crucial roles in all molecular mechanisms underlying the response to environmental challenges. In plants, different IDPs involved in stress response have been identified and characterized. Nevertheless, a comprehensive evaluation of protein disorder in plant proteomes under abiotic or biotic stresses is not available so far. In the present work the transcriptome dataset of strawberry (Fragaria X ananassa) fruits interacting with the fungal pathogen Colletotrichum acutatum was actualized onto the woodland strawberry (Fragaria vesca) genome. The obtained cDNA sequences were translated into protein sequences, which were subsequently subjected to disorder analysis. The results, providing the first estimation of disorder abundance associated to plant infection, showed that the proteome activated in the strawberry red fruit during the active fungal propagation is remarkably depleted in disorder. On the other hand, in the resistant white fruit, no significant disorder reduction is observed in the proteins expressed in response to fungal infection. Four representative proteins, FvSMP, FvPRKRIP, FvPCD-4 and FvFAM32A-like, predicted as mainly disordered and never experimentally characterized before, were isolated, and the absence of structure was validated at the secondary and tertiary level using circular dichroism and differential scanning fluorimetry. Their quaternary structure was also established using light scattering. The results are discussed considering the role of protein disorder in plant defense.


Assuntos
Colletotrichum/fisiologia , Fragaria/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Intrinsicamente Desordenadas/fisiologia , Doenças das Plantas/microbiologia , Fragaria/genética , Fragaria/fisiologia , Genes de Plantas/genética , Genes de Plantas/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia
18.
J Biol Inorg Chem ; 20(6): 1021-37, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26204982

RESUMO

Helicobacter pylori (Hp) is a carcinogen that relies on Ni(II) to survive in the extreme pH conditions of the human guts. The regulation of genes coding for Ni(II) enzymes and proteins is effected by the nickel-responsive transcription factor NikR, composed of a DNA-binding domain (DBD) and a metal-binding domain (MBD). The scope of this study is to obtain the molecular details of the HpNikR interaction with the urease operator OP ureA , in solution. The size of the full-length protein prevents the characterization of the HpNikR-OP ureA interaction using NMR. We thus investigated the two separate domains of HpNikR. The conservation of their oligomeric state was established by multiple-angle light scattering. Isothermal calorimetric titrations indicated that the thermodynamics of Ni(II) binding to the isolated MBD is independent of the presence of the adjacent DBDs. The NMR spectra of the isolated DBD support considerable conservation of its structural properties. The spectral perturbations induced on the DBD by OP ureA provided information useful to calculate a structural model of the HpNikR-OP ureA complex using a docking computational protocol. The NMR assignment of the residues involved in the protein-DNA interaction represents a starting point for the development of drugs potentially able to eradicate H. pylori infections. All evidences so far collected, in this and previous studies, consistently indicate that binding of Ni(II) to the MBD increases the HpNikR-DNA affinity by modulating the dynamic, and not the structural, properties of the protein, suggesting that the formation of a stable complex relies upon an induced fit mechanism.


Assuntos
Proteínas de Bactérias/genética , Regiões Operadoras Genéticas/genética , Proteínas Repressoras/genética , Helicobacter pylori , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Níquel , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/genética , Urease/genética
19.
Metallomics ; 7(9): 1305-18, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26099858

RESUMO

Nickel is an essential micronutrient for a large number of living organisms, but it is also a toxic metal ion when it accumulates beyond the sustainable level as it may result if and when its cellular trafficking is not properly governed. Therefore, the homeostasis and metabolism of nickel is tightly regulated through metal-specific protein networks that respond to the available Ni(II) concentration. These are directed by specific nickel sensors, able to couple Ni(II) binding to a change in their DNA binding affinity and/or specificity, thus translating the cellular level of Ni(II) into a modification of the expression of the proteins devoted to modulating nickel uptake, efflux and cellular utilization. This review describes the Ni(II)-dependent transcriptional regulators discovered so far, focusing on their structural features, metal coordination modes and metal binding thermodynamics. Understanding these properties is essential to comprehend how these sensors correlate nickel availability to metal coordination and functional responses. A broad and comparative study, described here, reveals some general traits that characterize the binding stoichiometry and Ni(II) affinity of these metallo-sensors.


Assuntos
Metaloproteínas/química , Metaloproteínas/metabolismo , Níquel/química , Níquel/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Termodinâmica
20.
J Biol Inorg Chem ; 20(4): 739-55, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25846143

RESUMO

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG.


Assuntos
Archaea/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Níquel/metabolismo , Urease/metabolismo , Zinco/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas Intrinsicamente Desordenadas/química , Mathanococcus/enzimologia , Proteínas de Ligação a Fosfato , Dobramento de Proteína , Temperatura Ambiente , Urease/química
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