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1.
Biochem Biophys Res Commun ; 615: 63-69, 2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35605407

RESUMO

HucR is a MarR family protein of Deinococcus radiodurans, which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding.


Assuntos
Deinococcus , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/química , Deinococcus/química , Ligação Proteica , Ácido Úrico/metabolismo , Água/metabolismo
2.
Phys Chem Chem Phys ; 24(9): 5394-5403, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35169823

RESUMO

Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα), have shown that quantifying the change in the denaturing temperature leads to some results that are unique to each protein. In order to find osmolyte-dependent parameters that can be applied more consistently from protein to protein, this work considers, instead, the overall free energy change associated with that denaturation using coarse-grained models. This is enabled by using theoretical fluid equations that take into account the exclusion of water and osmolyte from the volume occupied by the protein in both its native and denatured forms. Assuming ideal geometric models of the two protein states whose sizes are based on the protein's surface area in each form, and taking into account the density of the aqueous osmolyte solution, the free energy change due to the change in geometry can be calculated. The overall change in free energy of the system is found from that quantity and other protein- and osmolyte-specific parameters, which are determined using the experimental concentration and temperature results. We find that these fitted parameters accurately reproduce experimental results and also show consistent patterns from protein to protein. We also consider two different model geometries of the denatured protein and find little impact on the use of one or the other. Defining the effects of the osmolyte in terms of free energy also allows for prediction of overall phase change behavior, including cold denaturation.


Assuntos
Proteínas , Concentração Osmolar , Desnaturação Proteica , Proteínas/metabolismo , Temperatura , Termodinâmica
3.
Bioorg Med Chem Lett ; 47: 128216, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34157390

RESUMO

Malaria remains one of the deadliest infectious diseases worldwide and continues to infect hundreds of millions of individuals each year. Here we report the discovery and derivatization of a series of 2,6-dibenzylidenecyclohexanones targeting the chloroquine-sensitive 3D7 strain of Plasmodium falciparum . While the initial lead compound displayed significant toxicity in a human cell proliferation assay, we were able to identify a derivative with no detectable toxicity and sub-micromolar potency.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/síntese química , Antimaláricos/química , Proliferação de Células/efeitos dos fármacos , Cloroquina/síntese química , Cloroquina/química , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
4.
J Med Chem ; 63(20): 11902-11919, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-32945666

RESUMO

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade
5.
Proc Natl Acad Sci U S A ; 107(15): 6777-81, 2010 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-20351259

RESUMO

Many archaea (including all the methanogens, nearly all euryarchaeotes, and some crenarchaeotes) use histones as components of the chromatin that compacts their genomes. The archaeal histones are homo- and heterodimers that pair on DNA to form tetrasomes (as the eukaryotic histones H3 and H4 do). The resulting DNA packaging is known to interfere with assembly of the archaeal transcription apparatus at promoters; the ability of transcriptional activation to function in repressive archaeal chromatin has not yet been explored in vitro. Using four of the Methanocaldococcus jannaschii (Mja) histones, we have examined activation of the model Mja rb2 transcription unit by the Mja transcriptional activator Ptr2 in this simplified-chromatin context. Using hydroxyl radical footprinting, we find that the Ptr2-specific rb2 upstream activating site is a preferred histone-localizing site that nucleates histone: DNA-binding radiating from the rb2 promoter. Nevertheless, Ptr2 competes effectively with histones for access to the rb2 promoter and most potently activates transcription in vitro at histone concentrations that extensively coat DNA and essentially silence basal transcription.


Assuntos
Archaea/genética , Archaea/metabolismo , Histonas/química , Ativação Transcricional , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cromatina/química , DNA/química , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Genes Arqueais , Radical Hidroxila , Cinética , Modelos Genéticos , Regiões Promotoras Genéticas , Transcrição Gênica
6.
Mol Microbiol ; 74(3): 582-93, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19775246

RESUMO

Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA-binding proteins, is an activator of its eukaryal-type core transcription apparatus. In Lrp-family proteins, an N-terminal helix-turn-helix DNA-binding and dimerizing domain is joined to a C-terminal effector and multimerizing domain. A cysteine-scanning surface mutagenesis shows that the C-terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding-positive but activation-defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post-recruitment step. Transcriptional activation through the C-terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N-terminal DNA-binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr-H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr-H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr-H10 is a dimer; unlike Ptr2, the octameric Ptr-H16 poorly recruits TBP to the promoter, but more effectively co-recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Transativadores/química , Ativação Transcricional , Animais , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação/genética , Sequência Conservada/genética , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica em Archaea , Sequências Hélice-Volta-Hélice/genética , Proteína Reguladora de Resposta a Leucina/química , Proteína Reguladora de Resposta a Leucina/genética , Methanococcaceae/genética , Methanococcaceae/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Conformação Proteica , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo
7.
J Mol Biol ; 390(5): 1019-29, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19501097

RESUMO

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Cinética , Ligantes , Modelos Moleculares , Proteínas Mutantes/metabolismo , Regiões Operadoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Transcrição Gênica/efeitos dos fármacos , Ácido Úrico/química , Ácido Úrico/farmacologia , Xantina/química , Xantina/farmacologia
8.
J Mol Biol ; 360(1): 168-77, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16750221

RESUMO

We report here the 2.3 A resolution structure of the hypothetical uricase regulator (HucR) from Deinococcus radiodurans R1. HucR, a member of the MarR family of DNA-binding proteins, was previously shown to repress its own expression as well as that of a uricase, a repression that is alleviated both in vivo and in vitro upon binding uric acid, the substrate for uricase. As uric acid is a potent scavenger of reactive oxygen species, and as D. radiodurans is known for its remarkable resistance to DNA-damaging agents, these observations indicate a novel oxidative stress response mechanism. The crystal structure of HucR in the absence of ligand or DNA reveals a dimer in which the DNA recognition helices are preconfigured for DNA binding. This configuration of DNA-binding domains is achieved through an apparently stable dimer interface that, in contrast to what is observed in other MarR homologs for which structures have been determined, shows little conformational heterogeneity in the absence of ligand. An additional amino-terminal segment, absent from other MarR homologs, appears to brace the principal helix of the dimerization interface. However, although HucR is preconfigured for DNA binding, the presence of a stacked pair of symmetry-related histidine residues at a central pivot point in the dimer interface suggests a mechanism for a conformational change to attenuate DNA binding.


Assuntos
Proteínas de Bactérias/química , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química
9.
Curr Issues Mol Biol ; 8(1): 51-62, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16450885

RESUMO

The MarR (multiple antibiotic resistance regulator) family of prokaryotic transcriptional regulators includes proteins critical for control of virulence factor production, bacterial response to antibiotic and oxidative stresses and catabolism of environmental aromatic compounds. Recognition of the adaptive cellular responses mediated by MarR homologs, and the clinical isolation of antibiotic-resistant bacterial strains harboring MarR mutations, has garnered increasing medical and agricultural attention to this family. MarR proteins exist as homodimers in both free and DNA-bound states. Sequence specific DNA-binding to palindromic or pseudopalindromic sites is mediated by a conserved winged helix fold and, for numerous homologs, this association is attenuated by specific anionic lipophilic ligands. The mechanism of ligand-mediated allosteric control of DNA binding is unique amongst prokaryotic transcriptional regulators in that the DNA- and ligand-binding domains almost completely overlap in the residues involved. Until recently, our understanding of ligand-binding has been limited to a MarR-salicylate co-crystal structure, with little information on the allosteric mechanisms linking ligand-recognition and DNA-binding. However, recent biochemical and biophysical data on MarR homologs have begun to resolve the mechanisms by which these proteins mediate ligand-responsive transcriptional control.


Assuntos
Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Escherichia coli/genética , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína
10.
J Mol Biol ; 350(4): 617-30, 2005 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-15967460

RESUMO

Members of the MarR family of winged helix transcriptional regulators have been shown to regulate multidrug and oxidative stress response, pathogenesis, and catabolism of aromatic compounds. Many respond to anionic lipophilic compounds in their capacity to bind DNA, and the co-crystal structure of MarR bound to salicylate revealed two ligand-binding pockets, SAL-A and SAL-B. The MarR homolog, HucR, from Deinococcus radiodurans has been shown to repress expression of a predicted uricase, and DNA-binding by HucR is antagonized by uric acid, the substrate of uricase. We provide a biochemical investigation of DNA-binding and uric acid-binding by HucR. Equilibrium analytical ultracentrifugation indicates that HucR exists as a dimer. Intrinsic fluorescence spectra suggest that the association of the HucR dimer with its cognate DNA involves conformational flexibility in the globular interior and/or dimerization domain of the protein, and near-UV circular dichroism spectra indicate a concomitant change in the helical twist of the DNA duplex. DNA-binding affinity, measured by electrophoretic mobility-shift assays, for HucR mutants bearing single amino acid substitutions suggests the importance of the beta-hairpin "wing" in DNA binding. Analysis of intrinsic fluorescence spectra demonstrates that uric acid induces conformational changes in HucR and binds with an apparent K(d)=11.6(+/-3.7)muM and a Hill coefficient of 0.7+/-0.1, indicating negative cooperativity. Fluorescence and DNA-binding properties of the HucR variants indicate that SAL-A is a low-affinity, uric acid-binding site and that negative cooperativity exists between homologous, high-affinity sites. The conservation of residues comprising site SAL-A suggests that it is a low-affinity, ligand-binding site in MarR homologs. Mechanistic considerations suggest that HucR is regulated by uric acid to maintain optimal cellular levels of this scavenger of free radicals in response to oxidative stress and DNA damage.


Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácido Úrico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dicroísmo Circular , DNA/metabolismo , Deinococcus/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Espectrometria de Fluorescência
11.
J Mol Biol ; 347(3): 495-508, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15755446

RESUMO

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D.radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(+/-0.1) degrees C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca(2+) (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Deinococcus/química , Ferritinas , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , DNA/metabolismo , Deinococcus/metabolismo , Dimerização , Ferro/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência
12.
J Biol Chem ; 279(49): 51442-50, 2004 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-15448166

RESUMO

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities. HucR binds as a homodimer to a single site within its promoter/operator region with Kd = 0.29 +/- 0.02 nM. The HucR binding site contains a pseudopalindromic sequence, composed of 8-bp half-sites separated by 2 bp. The location of the HucR binding site in the intergenic region between hucR and a putative uricase suggests a mechanism of simultaneous co-repression of these two genes. The substrate of uricase, uric acid, is an efficient antagonist of DNA binding, reducing HucR-DNA complex formation to 50% at 0.26 mM ligand, compared with 5.2 and 46 mM for the aromatic compounds salicylate and acetylsalicylate, respectively. Enhanced levels in vivo of hucR and uricase transcript and increased uricase activity under conditions of excess uric acid further indicate a novel regulatory mechanism of aromatic catabolism in D. radiodurans. Since uric acid is a scavenger of reactive oxygen species, we hypothesize that HucR is a participant in the intrinsic resistance of D. radiodurans to high levels of oxidative stress.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Transcrição Gênica , Ácido Úrico/metabolismo , Sequência de Aminoácidos , Aspirina/química , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , DNA/química , DNA/metabolismo , DNA Intergênico , Deinococcus/metabolismo , Desoxirribonuclease I/química , Relação Dose-Resposta a Droga , Ácido Edético/química , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica , Cinética , Ligantes , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Fases de Leitura Aberta , Estresse Oxidativo , Fenol/química , Regiões Promotoras Genéticas , Ligação Proteica , RNA/química , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Repressoras/genética , Ácido Salicílico/química , Homologia de Sequência de Aminoácidos , Temperatura , Urato Oxidase/metabolismo
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