RESUMO
African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Proteínas Virais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/enzimologia , Animais , Suínos , Febre Suína Africana/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/química , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Ligação Proteica , DNA Viral/genética , DNA Viral/metabolismo , Cristalografia por Raios XRESUMO
Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.
Assuntos
Carbono , Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Homeostase , Ferro , Infecções Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Ferro/metabolismo , Carbono/metabolismo , Ácido Cítrico/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico , Camundongos , Transdução de SinaisRESUMO
U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.
Assuntos
Archaeoglobus , Modelos Moleculares , Fosfotransferases , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Sítios de Ligação , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Archaeoglobus/enzimologia , Fosfotransferases/químicaRESUMO
CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Humanos , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/química , Genoma , Endonucleases/genética , Endonucleases/metabolismo , Edição de GenesRESUMO
Chemo-activation of mitochondrial ClpP exhibits promising anticancer properties. However, we are currently unaware of any studies using selective and potent ClpP activators in lung squamous cell carcinoma. In this work, we report on such an activator, ZK53, which exhibits therapeutic effects on lung squamous cell carcinoma in vivo. The crystal structure of ZK53/ClpP complex reveals a π-π stacking effect that is essential for ligand binding selectively to the mitochondrial ClpP. ZK53 features on a simple scaffold, which is distinct from the activators with rigid scaffolds, such as acyldepsipeptides and imipridones. ZK53 treatment causes a decrease of the electron transport chain in a ClpP-dependent manner, which results in declined oxidative phosphorylation and ATP production in lung tumor cells. Mechanistically, ZK53 inhibits the adenoviral early region 2 binding factor targets and activates the ataxia-telangiectasia mutated-mediated DNA damage response, eventually triggering cell cycle arrest. Lastly, ZK53 exhibits therapeutic effects on lung squamous cell carcinoma cells in xenograft and autochthonous mouse models.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animais , Camundongos , Humanos , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/patologia , Endopeptidase Clp/metabolismoRESUMO
For shade-intolerant species, shade light indicates the close proximity of neighboring plants and triggers the shade avoidance syndrome (SAS), which causes exaggerated growth and reduced crop yield. Here, we report that non-secreted ROT FOUR LIKE (RTFL)/DEVIL (DVL) peptides negatively regulate SAS by interacting with BRASSINOSTEROID SIGNALING KINASEs (BSKs) and reducing the protein level of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) in Arabidopsis. The transcription of at least five RTFLs (RTFL13/16/17/18/21) is induced by low R:FR light. The RTFL18 (DVL1) protein is stabilized under low R:FR conditions and localized to the plasma membrane. A phenotype analysis reveals that RTFL18 negatively regulates low R:FR-promoted petiole elongation. BSK3 and BSK6 are identified as partners of RTFL18 through binding assays and structural modeling. The overexpression of RTFL18 or knockdown of BSK3/6 reduces BRASSINOSTEROID signaling and reduces low R:FR-stabilized PIF4 levels. Genetically, the overexpression of BSK3/6 and PIF4 restores the petiole phenotype acquired by RTFL18-overexpressing lines. Collectively, our work characterizes a signaling cascade (the RTFLs-BSK3/6-PIF4 pathway) that prevents the excessive activation of the shade avoidance response in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Transdução de Sinais , Fitocromo/metabolismo , Peptídeos/metabolismo , Regulação da Expressão Gênica de Plantas , LuzRESUMO
The fat mass and obesity-associated protein (FTO) is an RNA N6-methyladenosine (m6A) demethylase highly expressed in diverse cancers including acute myeloid leukemia (AML). To improve antileukemia drug-like properties, we have designed 44/ZLD115, a flexible alkaline side-chain-substituted benzoic acid FTO inhibitor derived from FB23. A combination of structure-activity relationship analysis and lipophilic efficiency-guided optimization demonstrates that 44/ZLD115 exhibits better drug-likeness than the previously reported FTO inhibitors, FB23 and 13a/Dac85. Then, 44/ZLD115 shows significant antiproliferative activity in leukemic NB4 and MOLM13 cell lines. Moreover, 44/ZLD115 treatment noticeably increases m6A abundance on the AML cell RNA, upregulates RARA gene expression, and downregulates MYC gene expression in MOLM13 cells, which are consistent with FTO gene knockdown. Lastly, 44/ZLD115 exhibits antileukemic activity in xenograft mice without substantial side effects. This FTO inhibitor demonstrates promising properties that can be further developed for antileukemia applications.
Assuntos
Leucemia Mieloide Aguda , RNA , Humanos , Camundongos , Animais , Linhagem Celular , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Relação Estrutura-Atividade , Genes myc , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genéticaRESUMO
Human caseinolytic protease P (ClpP) is required for the regulatory hydrolysis of mitochondrial proteins. Allosteric ClpP agonists dysfunctionally activate mitochondrial ClpP in antileukemic therapies. We previously developed ZG111, a potent ClpP agonist derived from ICG-001, inhibits the proliferation of pancreatic ductal adenocarcinoma cell lines in vitro and in vivo by degrading respiratory chain complex proteins. Herein, we studied the structure-activity relationships of ICG-001 analogs as antileukemia agents. Compound ZG36 exhibited improved stabilization effects on the thermal stability of ClpP in acute myeloid leukemia (AML) cell lines compared with the stabilization effects of ZG111, indicating a direct binding between ZG36 and ClpP. Indeed, the resolved ZG36/ClpP structural complex reveals the mode of action of ZG36 during ClpP binding. Compound ZG36 nonselectively degrades respiratory chain complexes and decreases the mitochondrial DNA, eventually leading to the collapse of mitochondrial function and leukemic cell death. Finally, ZG36 treatment inhibited 3-D cell growth in vitro and suppressed the tumorigenesis of AML cells in xenografted mice models. Collectively, we developed a new class of human ClpP agonists that can be used as potential antileukemic therapies.
Assuntos
Leucemia Mieloide Aguda , Mitocôndrias , Animais , Humanos , Camundongos , Linhagem Celular , Endopeptidase Clp/química , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mitocôndrias/metabolismo , Relação Estrutura-AtividadeRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tuâ¢EF-Ts and EF-Tuâ¢GDP complexes, showing the molecular basis of EF-Tu's representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.
Assuntos
Mycobacterium tuberculosis , Fator Tu de Elongação de Peptídeos , Vacina BCG , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Infection of human parvovirus B19 (B19V) can cause a variety of diseases, such as hydrops fetalis, erythema infectiosum in children and acute arthropathy in women. Although B19V infection mainly occurs during childhood, about 50 % of adults are still susceptible to B19V infection. As the major replication protein of B19V, deletion of NS1 completely abolishes the infectivity of the virus. The nuclease domain of NS1 (NS1_Nuc) is responsible for DNA Ori binding and nicking that is critical for B19V viral DNA replication. NS1 has various variants, the structure and function for the majority of the variants are poorly studied. Here, we report two high-resolution crystal structures of NS1_Nuc, revealed the detailed conformations of many key residues. Structural comparison indicates that these residues are important for ssDNA or dsDNA binding by NS1. NS1 belongs to the HUH-endonuclease superfamily and it shares conserved ssDNA cleavage mechanism with other HUH-endonuclease members. However, our structural analyses, mutagenesis and in vitro assay results all suggested that NS1_Nuc utilizes one unique model in ssDNA binding.
RESUMO
The N6-methyladenosine (m6A) demethylase FTO is overexpressed in acute myeloid leukemia (AML) cells and promotes leukemogenesis. We previously developed tricyclic benzoic acid FB23 as a highly potent FTO inhibitor in vitro. However, it showed a moderate antiproliferative effect on AML cells. In this work, we performed a structure-activity relationship study of tricyclic benzoic acids as FTO inhibitors. The analog 13a exhibited excellent inhibitory effects on FTO similar to that of FB23 in vitro. In contrast to FB23, 13a exerted a strong antiproliferative effect on AML cells. Like FTO knock down, 13a upregulated ASB2 and RARA expression and increased the protein abundance while it downregulated MYC expression and decreased MYC protein abundance. These genes are key FTO targets in AML cells. Finally, 13a treatment improved the survival rate of MONOMAC6-transplanted NSG mice. Collectively, our data suggest that targeting FTO with tricyclic benzoic acid inhibitors may be a potential strategy for treating AML.
Assuntos
Ácido Benzoico , Leucemia Mieloide Aguda , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Ácido Benzoico/farmacologia , Carcinogênese , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Relação Estrutura-AtividadeRESUMO
The mitochondrial caseinolytic protease P (ClpP) is a target candidate for treating leukemia; however, the effects of ClpP modulation on solid tumors have not been adequately explored. Here, we report a potent activator of ClpP with the therapeutic potential for pancreatic ductal adenocarcinoma (PDAC). We first validated that aberrant ClpP activation leads to growth arrest of PDAC cells and tumors. We then performed high-throughput screening and synthetic optimization, from which we identified ZG111, a potent activator of ClpP. ZG111 binds to ClpP and promotes the ClpP-mediated degradation of respiratory chain complexes. This degradation activates the JNK/c-Jun pathway, induces the endoplasmic reticulum stress response, and consequently causes the growth arrest of PDAC cells. ZG111 also produces inhibitory effects on tumor growth in cell line-derived and patient-derived xenograft mouse models. Altogether, our data demonstrate a promising therapeutic strategy for PDAC suppression through the chemical activation of ClpP.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/metabolismo , Endopeptidase Clp/metabolismo , Homeostase , Humanos , Camundongos , Neoplasias Pancreáticas/metabolismo , Peptídeo Hidrolases/metabolismo , Proteoma/metabolismo , Neoplasias PancreáticasRESUMO
The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.
RESUMO
Detoxification of the highly toxic cadmium element is essential for the survival of living organisms. Pseudomonas putida CadR, a MerR family transcriptional regulator, has been reported to exhibit an ultraspecific response to the cadmium ion. Our crystallographic and spectroscopic studies reveal that the extra cadmium selectivity of CadR is mediated by the unexpected cooperation of thiolate-rich site I and histidine-rich site II. Cadmium binding in site I mediates the reorientation of protein domains and facilitates the assembly of site II. Subsequently, site II bridge-links 2 DNA binding domains through ligands His140/His145 in the C-terminal histidine-rich tail. With dynamic transit between 2 conformational states, this bridge could stabilize the regulator into an optimal conformation that is critical for enhancing the transcriptional activity of the cadmium detoxification system. Our results provide dynamic insight into how nature utilizes the unique cooperative binding mechanism in multisite proteins to recognize cadmium ions specifically.
Assuntos
Proteínas de Bactérias/metabolismo , Cádmio/metabolismo , Pseudomonas putida/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Chumbo/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Pseudomonas putida/genética , Fatores de Transcrição/genética , Zinco/metabolismoRESUMO
FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Dioxigenase FTO Dependente de alfa-Cetoglutarato/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Metilação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Terapia de Alvo Molecular , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Relação Estrutura-Atividade , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DUX4 plays critical role in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy and acute lymphoblastic leukemia in humans. As a master transcription regulator, DUX4 can also bind the promoters and activate the transcription of hundreds ZGA-associated genes. Here we report on the structural and biochemical studies of DUX4 double homeodomains (DUX4-DH), representing the only structures contain both homeodomain 1 (HD1) and homeodomain 2 (HD2). HD1 and HD2 adopt classical homeobox fold; via the helix inserted into the major groove and the N-terminal extended loop inserted into the minor groove, HD1 and HD2 recognize the box1 (5'-TAA-3') and box2 (5'-TGA-3') nucleotides of the consensus sequence, respectively. Among the box1 and box2 linking nucleotides (CCTAA), the two adenine residues are reported to be highly conserved; however, they are not directly recognized by DUX4-DH in the structures. Besides different nucleotides, our ITC analysis indicated that DUX4-DH can also tolerate various changes in the linker length. Our studies not only revealed the basis for target DNA recognition by DUX4, but also advanced our understanding on multiple gene activation by DUX4.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Adenina/química , DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Mutagênese , Motivos de Nucleotídeos , Nucleotídeos/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Recent findings have revealed that the protein gasdermin D (GSDMD) plays key roles in cell pyroptosis. GSDMD binds lipids and forms pore structures to induce pyroptosis upon microbial infection and associated danger signals. However, detailed structural information for GSDMD remains unknown. Here, we report the crystal structure of the C-terminal domain of human GSDMD (GSDMD-C) at 2.64-Å resolution. The first loop on GSDMD-C inserts into the N-terminal domain (GSDMD-N), which helps stabilize the conformation of the full-length GSDMD. Substitution of this region by a short linker sequence increased levels of cell death. Mutants F283A and F283R can increase protein heterogeneity in vitro and are capable of undergoing cell pyroptosis in 293T cells. The small-angle X-ray-scattering envelope of human GSDMD is consistent with the modeled GSDMD structure and mouse GSDMA3 structure, which suggests that GSDMD adopts an autoinhibited conformation in solution. The positive potential surface of GSDMD-N covered by GSDMD-C is exposed after being released from the autoinhibition state and can form high-order oligomers via a charge-charge interaction. Furthermore, by mapping different regions of GSDMD, we determined that one short segment is sufficient to kill bacteria in vitro and can efficiently inhibit cell growth in Escherichia coli and Mycobacterium Smegmatis These findings reveal that GSDMD-C acts as an auto-inhibition executor and GSDMD-N could form pore structures via a charge-charge interaction upon cleavage by caspases during cell pyroptosis.
Assuntos
Modelos Moleculares , Proteínas de Neoplasias/química , Piroptose , Substituição de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Cristalografia por Raios X , Escherichia coli/crescimento & desenvolvimento , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Mutação de Sentido Incorreto , Mycobacterium smegmatis/crescimento & desenvolvimento , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Proteínas de Ligação a Fosfato , Domínios Proteicos , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMO
As a master redox-sensing MarR-family transcriptional regulator, AbfR participates in oxidative stress responses and virulence regulations in Staphylococcus epidermidis. Here, we present structural insights into the DNA-binding mechanism of AbfR in different oxidation states by determining the X-ray crystal structures of a reduced-AbfR/DNA complex, an overoxidized (Cys13-SO2H and Cys13-SO3H) AbfR/DNA, and 2-disulfide cross-linked AbfR dimer. Together with biochemical analyses, our results suggest that the redox regulation of AbfR-sensing displays two novel features: (i) the reversible disulfide modification, but not the irreversible overoxidation, significantly abolishes the DNA-binding ability of the AbfR repressor; (ii) either 1-disulfide cross-linked or 2-disulfide cross-linked AbfR dimer is biologically significant. The overoxidized species of AbfR, resembling the reduced AbfR in conformation and retaining the DNA-binding ability, does not exist in biologically significant concentrations, however. The 1-disulfide cross-linked modification endows AbfR with significantly weakened capability for DNA-binding. The 2-disulfide cross-linked AbfR adopts a very "open" conformation that is incompatible with DNA-binding. Overall, the concise oxidation chemistry of the redox-active cysteine allows AbfR to sense and respond to oxidative stress correctly and efficiently.
Assuntos
DNA/metabolismo , Staphylococcus epidermidis/metabolismo , Fatores de Transcrição/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , DNA/química , Dissulfetos/química , Dissulfetos/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Fatores de Transcrição/químicaRESUMO
The transcription regulator PbrR691, one of the MerR family proteins, shows extremely high sensitivity and selectivity toward Pb(II) in Ralstonia metallidurans CH34. Here, we present the crystal structure of PbrR691 in complex with Pb(II) at 2.0 Å resolution. The Pb(II) coordinates with three conserved cysteines and adopts a unique trigonal-pyramidal (hemidirected) geometry. To our knowledge, the PbrR691-Pb(II) structure provides the first three-dimensional visualization of a functional hemidirected lead(II) thiolate coordinate geometry in a protein.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Chumbo/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Estrutura Molecular , Fatores de Transcrição/químicaRESUMO
The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.