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1.
J Med Microbiol ; 70(4)2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33830911

RESUMEN

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa.Methodology. Computer-based virtual screening under consideration of the 'eNTRy' rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus.Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.


Asunto(s)
Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Transactivadores/antagonistas & inhibidores , Animales , Drosophila , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Simulación del Acoplamiento Molecular , Mutación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/mortalidad , Pseudomonas aeruginosa/genética , Tasa de Supervivencia , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Virulencia/efectos de los fármacos , Virulencia/genética
2.
Nat Commun ; 12(1): 1846, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758180

RESUMEN

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.


Asunto(s)
Evolución Molecular Dirigida/métodos , Genes de Cambio , Ingeniería Genética/métodos , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Citometría de Flujo , Biblioteca de Genes , Genes Reporteros , Floroglucinol/análogos & derivados , Floroglucinol/farmacología , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Relación Señal-Ruido , Tetraciclina/farmacología , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , beta Caroteno/biosíntesis , beta Caroteno/genética , beta Caroteno/metabolismo
3.
Nucleic Acids Res ; 49(4): 2255-2265, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33533913

RESUMEN

Interferon regulatory factor 4 (IRF4) is a key transcription factor (TF) in the regulation of immune cells, including B and T cells. It acts by binding DNA as both a homodimer and, in conjunction with other TFs, as a heterodimer. The choice of homo and heterodimeric/ DNA interactions is a critical aspect in the control of the transcriptional program and cell fate outcome. To characterize the nature of this interaction in the homodimeric complex, we have determined the crystal structure of the IRF4/ISRE homodimeric complex. We show that the complex formation is aided by a substantial DNA deformation with co-operative binding achieved exclusively through protein-DNA contact. This markedly contrasts with the heterodimeric form where DNA bound IRF4 is shown to physically interact with PU.1 TF to engage EICE1. We also show that the hotspot residues (Arg98, Cys99 and Asn102) contact both consensus and non-consensus sequences with the L1 loop exhibiting marked flexibility. Additionally, we identified that IRF4L116R, a mutant associated with chronic lymphocytic leukemia, binds more robustly to DNA thereby providing a rationale for the observed gain of function. Together, we demonstrate key structural differences between IRF4 homo and heterodimeric complexes, thereby providing molecular insights into IRF4-mediated transcriptional regulation.


Asunto(s)
ADN/química , Factores Reguladores del Interferón/química , ADN/metabolismo , Dimerización , Mutación con Ganancia de Función , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas Proto-Oncogénicas/química , Transactivadores/química
4.
Nucleic Acids Res ; 49(2): 1046-1064, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33410911

RESUMEN

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.


Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Ingeniería de Proteínas/métodos , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Circoviridae/enzimología , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/química , ADN de Cadena Simple/química , Desoxirribonucleasa I/química , Biblioteca de Genes , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Virus de Plantas/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/química , Especificidad por Sustrato , Transactivadores/química , Proteínas Virales/química
5.
Biochim Biophys Acta Gen Subj ; 1865(3): 129810, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33309686

RESUMEN

BACKGROUND: Zra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn2+, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. METHODS: The purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. RESULTS: We show that ZraS binds one Zn2+ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. CONCLUSION: Our results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. GENERAL SIGNIFICANCE: We bring new input on the so far poorly described Zra system and notably on ZraS.


Asunto(s)
Arabinosa/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Transactivadores/química , Zinc/química , Secuencia de Aminoácidos , Arabinosa/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Periplasma/genética , Periplasma/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Transactivadores/genética , Transactivadores/metabolismo , Zinc/metabolismo
6.
Infect Immun ; 88(11)2020 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-32817331

RESUMEN

Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.


Asunto(s)
Proteínas Bacterianas/genética , Fascitis Necrotizante/microbiología , Histidina Quinasa/genética , Miositis/microbiología , Proteínas Represoras/genética , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Transactivadores/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fascitis Necrotizante/metabolismo , Fascitis Necrotizante/mortalidad , Fascitis Necrotizante/patología , Femenino , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina Quinasa/química , Histidina Quinasa/metabolismo , Humanos , Ratones , Mutación , Miositis/metabolismo , Miositis/mortalidad , Miositis/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/mortalidad , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidad , Análisis de Supervivencia , Transactivadores/química , Transactivadores/metabolismo , Virulencia
7.
Nucleic Acids Res ; 48(14): 8113-8127, 2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32658272

RESUMEN

Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Multimerización de Proteína , Transactivadores/metabolismo , Bacillus subtilis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conjugación Genética/genética , Péptidos/metabolismo , Regiones Promotoras Genéticas , Repeticiones de Tetratricopéptidos , Transactivadores/química , Transactivadores/genética
8.
Avian Dis ; 64(2): 174-182, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32550618

RESUMEN

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus type 2. Gallid alphaherpesvirus type 3 (GaHV-3) strain 301B/1 was previously shown to be an effective MD vaccine with synergistic efficacy when used as a bivalent vaccine with turkey herpesvirus. Since the nucleotide sequences of only two GaHV-3 strains have been determined, we sought to sequence the 301B/1 genome using Illumina MiSeq technology. Phylogenomic analysis indicated that 301B/1 is more closely related to other GaHV-3 strains (SB-1 and HPRS24) than to virulent or attenuated strains of GaHV-2. One hundred and twenty-six open reading frames (ORFs) have been identified within the 301B/1 genome with 108 ORFs showing a high degree of similarity to homologs found in the genomes of SB-1 and HPRS24; 14 ORFs are highly homologous (> 90% identity) with the corresponding ORFs within the SB-1 genome. The R-LORF8 and R-LORF9 genes are the most dissimilar to the collinear genes found in the SB-1 genome but are highly homologous (99%-100% identity) with those within the HPRS24 genome. Overall the 301B/1 genome is most similar to the SB-1 virus genome (99.1%) and to a lesser degree with the HPRS24 virus genome (97.7%). However, six 301B/1 ORFs (UL47, UL48, UL52, pp38, ICP4, and US10) have been identified that contain nonsynonymous substitutions relative to homologs found in the SB-1 genome. Notably, unlike the avian retrovirus long terminal repeat sequences found within the SB-1 genome, none were identified within the 301B/1 genome.


Asunto(s)
Antígenos Virales/genética , Herpesvirus Gallináceo 3/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Transactivadores/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Antígenos Virales/metabolismo , Embrión de Pollo , Pollos , Enfermedad de Marek/virología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Enfermedades de las Aves de Corral/virología , Alineación de Secuencia , Transactivadores/química , Transactivadores/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo
9.
Nature ; 583(7815): 310-313, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32494006

RESUMEN

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.


Asunto(s)
Microscopía por Crioelectrón , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/ultraestructura , Secuencia de Bases , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Transactivadores/química , Transactivadores/metabolismo
10.
Nat Commun ; 11(1): 2728, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32483114

RESUMEN

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.


Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Conformación Proteica , Multimerización de Proteína , Transactivadores/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenol/metabolismo , Unión Proteica , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo
11.
PLoS Genet ; 16(5): e1008818, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32469866

RESUMEN

The Hippo signalling pathway and its central effector YAP regulate proliferation of cardiomyocytes and growth of the heart. Using genetic models in mice we show that the increased proliferation of embryonal and postnatal cardiomyocytes due to loss of the Hippo-signaling component SAV1 depends on the Myb-MuvB (MMB) complex. Similarly, proliferation of postnatal cardiomyocytes induced by constitutive active YAP requires MMB. Genome studies revealed that YAP and MMB regulate an overlapping set of cell cycle genes in cardiomyocytes. Protein-protein interaction studies in cell lines and with recombinant proteins showed that YAP binds directly to B-MYB, a subunit of MMB, in a manner dependent on the YAP WW domains and a PPXY motif in B-MYB. Disruption of the interaction by overexpression of the YAP binding domain of B-MYB strongly inhibits the proliferation of cardiomyocytes. Our results point to MMB as a critical downstream effector of YAP in the control of cardiomyocyte proliferation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Miocitos Cardíacos/citología , Transactivadores/química , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proliferación Celular , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miocitos Cardíacos/química , Regiones Promotoras Genéticas , Ratas
12.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-32332134

RESUMEN

Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization.IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.


Asunto(s)
Proteínas Bacterianas/genética , Endófitos/genética , Enterobacteriaceae/genética , Proteínas Represoras/genética , Transactivadores/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endófitos/metabolismo , Enterobacteriaceae/metabolismo , Oryza/microbiología , Filogenia , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Alineación de Secuencia , Simbiosis/genética , Transactivadores/química , Transactivadores/metabolismo
13.
Phys Chem Chem Phys ; 22(15): 8118-8127, 2020 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-32242581

RESUMEN

Many intrinsically disordered proteins (IDPs) are involved in complex signalling networks inside the cell. Their particular binding modes elicit different types of responses that can be subtly regulated. Here we study the binding of two disordered transactivation domains from proteins HIF-1α and CITED2, whose binding to the TAZ1 domain of CBP is critical for the hypoxic response. Experiments have shown that both IDPs compete for their shared partner, and that this competition is mediated by the formation of a ternary intermediate state. Here we use computer simulations with a coarse-grained model to provide a detailed molecular description of this intermediate. We find that the conserved LP(Q/E)L motif may have a critical role in the displacement of HIF-1α by CITED2 and show a possible mechanism for the transition from the intermediate to the bound state. We also explore the role of TAZ1 dynamics in the binding. The results of our simulations are consistent with many of the experimental observations and provide a detailed view of the emergent properties in the complex binding of these IDPs.


Asunto(s)
Simulación por Computador , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Modelos Moleculares , Dominios Proteicos , Proteínas Represoras/química , Transactivadores/química , Secuencias de Aminoácidos , Unión Proteica , Estructura Cuaternaria de Proteína
14.
Sci Adv ; 6(8): eaay3178, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32128405

RESUMEN

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.


Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Multimerización de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , ADN/metabolismo , Retroalimentación Fisiológica , Humanos , Proteínas Intrínsecamente Desordenadas/química , Mutación/genética , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Espectroscopía de Protones por Resonancia Magnética , Electricidad Estática , Transactivadores/química , Transactivadores/genética , Activación Transcripcional
15.
Proc Natl Acad Sci U S A ; 117(11): 5595-5603, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32123067

RESUMEN

The TAZ1 domain of CREB binding protein is crucial for transcriptional regulation and recognizes multiple targets. The interactions between TAZ1 and its specific targets are related to the cellular hypoxic negative feedback regulation. Previous experiments reported that one of the TAZ1 targets, CITED2, is an efficient competitor of another target, HIF-1α. Here, by developing the structure-based models of TAZ1 complexes, we have uncovered the underlying mechanisms of the competitions between the two intrinsic disordered proteins (IDPs) HIF-1α and CITED2 binding to TAZ1. Our results support the experimental hypothesis on the competition mechanisms and the apparent affinity. Furthermore, the simulations locate the dominant position of forming TAZ1-CITED2 complex in both thermodynamics and kinetics. For thermodynamics, TAZ1-CITED2 is the lowest basin located on the free energy surface of binding in the ternary system. For kinetics, the results suggest that CITED2 binds to TAZ1 faster than HIF-1α. In addition, the analysis of contact map and Φ values is important for guiding further experimental studies to understand the biomolecular functions of IDPs.


Asunto(s)
Proteína de Unión a CREB/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Proteínas Intrínsecamente Desordenadas/química , Simulación de Dinámica Molecular , Proteínas Represoras/química , Transactivadores/química , Sitios de Unión , Proteína de Unión a CREB/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Transactivadores/metabolismo
16.
Dev Cell ; 52(5): 605-616.e7, 2020 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-32032548

RESUMEN

The expression of multiple growth-promoting genes is coordinated by the transcriptional co-activator Yorkie with its major regulatory input provided by the Hippo-Warts kinase cascade. Here, we identify Atg1/ULK1-mediated phosphorylation of Yorkie as an additional inhibitory input independent of the Hippo-Warts pathway. Two serine residues in Yorkie, S74 and S97, are Atg1/ULK1 consensus target sites and are phosphorylated by ULK1 in vitro, thereby preventing its binding to Scalloped. In vivo, gain of function of Atg1, or its activator Acinus, caused elevated Yorkie phosphorylation and inhibited Yorkie's growth-promoting activity. Loss of function of Atg1 or Acinus raised expression of Yorkie target genes and increased tissue size. Unlike Atg1's role in autophagy, Atg1-mediated phosphorylation of Yorkie does not require Atg13. Atg1 is activated by starvation and other cellular stressors and therefore can impose temporary stress-induced constraints on the growth-promoting gene networks under the control of Hippo-Yorkie signaling.


Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Sitios de Unión , Núcleo Celular/metabolismo , Ojo Compuesto de los Artrópodos/metabolismo , Secuencia de Consenso , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
17.
Nat Commun ; 11(1): 769, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32034139

RESUMEN

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Asunto(s)
Histidina Quinasa/química , Histidina Quinasa/metabolismo , Thermotoga maritima/enzimología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinasa/genética , Concentración de Iones de Hidrógeno , Modelos Biológicos , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mutación , Fosforilación , Conformación Proteica , Thermotoga maritima/genética , Transactivadores/química , Transactivadores/metabolismo
18.
Nature ; 577(7792): 717-720, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31969703

RESUMEN

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)1,2 and transcription factor IID (TFIID)2-4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7-9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10-14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15-17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.


Asunto(s)
Microscopía por Crioelectrón , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae , Transactivadores/química , Transactivadores/ultraestructura , Transcripción Genética , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/ultraestructura , Histonas/metabolismo , Modelos Moleculares , Nucleosomas/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/metabolismo , Factor de Transcripción TFIID/metabolismo , Ubiquitinación
19.
Nature ; 577(7792): 711-716, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31969704

RESUMEN

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.


Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pichia , Regiones Promotoras Genéticas/genética , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Pichia/química , Pichia/genética , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIIA/química , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo
20.
J Phys Chem Lett ; 11(3): 864-868, 2020 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-31940206

RESUMEN

The transcriptional adaptor zinc-binding 1 (TAZ1) domain of the transcriptional coactivator CBP/P300 and two disordered peptides, HIF-1α and CITED2, form a delicate protein switch that regulates cellular hypoxic response. In hypoxia, HIF-1α binds TAZ1 to control the transcription of adaptive genes critical for the recovery from hypoxic stress. CITED2 acts as the negative feedback regulator to rapidly displace HIF-1α and efficiently attenuate the hypoxic response. Though CITED2 and HIF-1α have the same dissociation constant (Kd = 10 nM) in their binary complexes with TAZ1, CITED2 is much more competitive than HIF-1α upon binding the same target TAZ1 in ternary ( Berlow et al. Nature 2017 , 543 , 447 - 451 ). Here we demonstrate that a simple coarse-grained model can recapitulate this negative allosteric effect and provide detailed physical insights into the displacement mechanism. We find that long-range electrostatic forces are essential for the efficient displacement of HIF-1α by CITED2. The strong electrostatic interactions between CITED2 and TAZ1, along with the unique binding mode, make CITED2 much more competitive than HIF-1α in binding TAZ1.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Proteínas Represoras/química , Transactivadores/química , Factores de Transcripción p300-CBP/química , Regulación Alostérica , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Electricidad Estática
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