RESUMEN
Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos , Sitios de Unión , Descubrimiento de Drogas , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Unión Proteica , Dominios Proteicos , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder (GAND). METHODS: Fifty GAND subjects were evaluated to determine consistent genotypic/phenotypic features. Immunoprecipitation assays utilizing in vitro transcription-translation products were used to evaluate GATAD2B missense variants' ability to interact with binding partners within the nucleosome remodeling and deacetylase (NuRD) complex. RESULTS: Subjects had clinical findings that included macrocephaly, hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios, apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified with multiple variant types (nonsense, truncating frameshift, splice-site variants, deletions, and missense). Seven subjects were identified with missense variants that localized within two conserved region domains (CR1 or CR2) of the GATAD2B protein. Immunoprecipitation assays revealed several of these missense variants disrupted GATAD2B interactions with its NuRD complex binding partners. CONCLUSIONS: A consistent GAND phenotype was caused by a range of genetic variants in GATAD2B that include loss-of-function and missense subtypes. Missense variants were present in conserved region domains that disrupted assembly of NuRD complex proteins. GAND's clinical phenotype had substantial clinical overlap with other disorders associated with the NuRD complex that involve CHD3 and CHD4, with clinical features of hypotonia, intellectual disability, cardiac defects, childhood apraxia of speech, and macrocephaly.
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Discapacidad Intelectual , Megalencefalia , Trastornos del Neurodesarrollo , Niño , Femenino , Factores de Transcripción GATA/genética , Humanos , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Nucleosomas , Fenotipo , Embarazo , Proteínas RepresorasRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined. In particular, the functions of the other domains such as the ET domain have been less extensively studied. Here, we examine the properties of the ET domain of BRD3 as a protein/protein interaction module. Using a combination of pulldown and biophysical assays, we demonstrate that BRD3 binds to a range of chromatin-remodeling complexes, including the NuRD, BAF, and INO80 complexes, via a short linear "KIKL" motif in one of the complex subunits. NMR-based structural analysis revealed that, surprisingly, this mode of interaction is shared by the AF9 and ENL transcriptional coregulators that contain an acetyl-lysine-binding YEATS domain and regulate transcriptional elongation. This observation establishes a functional commonality between these two families of cancer-related transcriptional regulators. In summary, our data provide insight into the mechanisms by which BET family proteins might link chromatin acetylation to transcriptional outcomes and uncover an unexpected functional similarity between BET and YEATS family proteins.
Asunto(s)
Ensamble y Desensamble de Cromatina , Péptidos/química , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Acetilación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fenómenos Biofísicos , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Redes Reguladoras de Genes , Células HEK293 , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de TranscripciónRESUMEN
Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which ß-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.
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ADN Intergénico/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Proteínas con Homeodominio LIM/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , ADN Intergénico/química , ADN Intergénico/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas con Homeodominio LIM/química , Proteínas con Homeodominio LIM/genética , Ratones , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Mutación/genética , Unión Proteica/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises â¼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.
Asunto(s)
Autoantígenos/metabolismo , ADN Helicasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Nucleosomas/metabolismo , Animales , Autoantígenos/genética , Línea Celular , ADN Helicasas/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Nucleosomas/genéticaRESUMEN
Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.
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Autoantígenos/química , Autoantígenos/metabolismo , Dominios HMG-Box , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Ensamble y Desensamble de Cromatina , Secuencia Conservada , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
This study investigated whether or not prepubertal exposure to the fish contaminants methylmercury (MeHg) and the polychlorinated bisphenol Aroclor in low doses interferes with the histomorphometry of the testes, epididymis, liver and kidneys in rats. Wistar male rats, 21 days old, were allocated into the following: control (n = 17, received corn oil), MeHg (n = 17, received MeHg at 0.5 mg/kg/day), Aroclor (n = 17, received Aroclor at 1.0 mg/kg/day), low mix (n = 18, received MeHg at 0.05 mg/kg/day and Aroclor at 0.1 mg/kg/day), high mix (n = 18, received MeHg at 0.5 mg/kg/day and Aroclor at 1.0 mg/kg/day). Dosing continued from post natal day (PND) 23 to 53, by gavage. Euthanasia was performed on PND 53; or, after an interval of 62 days without exposure to chemicals, on PND 115. The degree of maturation of the seminiferous epithelium was delayed in chemical-exposed groups and testicular interstitial oedema was observed at adulthood. The pattern of male gonad organization was changed in the Aroclor group on PND 53 and in all treated groups at adulthood. The animals from Aroclor, low mix and high mix groups showed a reduction in the number of Sertoli cells. Histological evidence of renal injury was observed in all chemical-exposed groups in both ages. A probable target for MeHg and Aroclor in the reproductive system was Sertoli cells, in which possible dysfunctions could be linked to the other testicular alterations. Curiously, the main deleterious effects were late outcomes, along with the absence of synergistic interaction of MeHg and Aroclor in the parameters investigated. In conclusion, fish pollutants MeHg and Aroclor caused permanent structural damage in male gonads and kidneys after prepubertal exposure, without showing clear chemical interactions.
Asunto(s)
Arocloros/toxicidad , Peces/metabolismo , Contaminación de Alimentos , Riñón/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Testículo/efectos de los fármacos , Animales , Biometría/métodos , Contaminantes Ambientales/toxicidad , Femenino , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Pubertad , Ratas Wistar , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/patología , Espermatogénesis/efectos de los fármacos , Testículo/patología , Aumento de Peso/efectos de los fármacosRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.
Asunto(s)
Histona Desacetilasas/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Proteínas Represoras/química , Proteína 4 de Unión a Retinoblastoma/química , Secuencia de Aminoácidos , Animales , Ensamble y Desensamble de Cromatina , Secuencia Conservada , Cristalografía por Rayos X , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Proteína 7 de Unión a Retinoblastoma/química , Proteína 7 de Unión a Retinoblastoma/genética , Proteína 7 de Unión a Retinoblastoma/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).
Asunto(s)
Marcadores de Afinidad , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/aislamiento & purificación , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Péptidos/química , Secuencia de Aminoácidos , Animales , Catálisis , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Ratones , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.
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Translocasas Mitocondriales de ADP y ATPRESUMEN
Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3'-azido-3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP-ADP and AZT-MP-ADP) and a binary complex with the transition state analogue AP5dT [P1-(5'-adenosyl)-P5-(5'-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.
Asunto(s)
Nucleótidos de Desoxiguanina/metabolismo , Didesoxinucleótidos/química , Didesoxinucleótidos/metabolismo , Nucleósido-Fosfato Quinasa/química , Plasmodium falciparum/enzimología , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Nucleótidos de Desoxiguanina/química , Cinética , Nucleósido-Fosfato Quinasa/metabolismo , Fosforilación , Plasmodium falciparum/metabolismo , Especificidad por Sustrato , Zidovudina/química , Zidovudina/metabolismoRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
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Regulación de la Expresión Génica/genética , Histona Desacetilasas/metabolismo , Nucleosomas/metabolismo , Humanos , Modelos MolecularesRESUMEN
MTH909 is the Methanothermobacter thermautotrophicus orthologue of Saccharomyces cerevisiae TAN1, which is required for N(4)-acetylcytidine formation in tRNA. The protein consists of an N-terminal near-ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein that is capable of making base modifications. MTH909 has been cloned, overexpressed and purified. The molecule exists as a monomer in solution. X-ray data were collected to 2.85 A resolution from a native crystal belonging to space group P6(1)22 (or P6(5)22), with unit-cell parameters a = 69.9, c = 408.5 A.
Asunto(s)
Proteínas Bacterianas/química , Methanobacteriaceae/química , Proteínas de Unión al ARN/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalización , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Difracción de Rayos XRESUMEN
Mycobacterium tuberculosis protein-tyrosine-phosphatase B (MptpB) is a secreted virulence factor that subverts antimicrobial activity in the host. We report here the structure-based design of selective MptpB inhibitors that reduce survival of multidrug-resistant tuberculosis strains in macrophages and enhance killing efficacy by first-line antibiotics. Monotherapy with an orally bioavailable MptpB inhibitor reduces infection burden in acute and chronic guinea pig models and improves the overall pathology. Our findings provide a new paradigm for tuberculosis treatment.
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Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Diseño de Fármacos , Macrófagos/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Animales , Proteínas Bacterianas/química , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Femenino , Cobayas , Macrófagos/microbiología , Macrófagos/patología , Masculino , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Proteínas Tirosina Fosfatasas/química , Relación Estructura-Actividad , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
We previously identified a series of inositol polyphosphate kinases (IPKs), Arg1, Ipk1, Kcs1 and Asp1, in the opportunistic fungal pathogen Cryptococcus neoformans. Using gene deletion analysis, we characterized Arg1, Ipk1 and Kcs1 and showed that they act sequentially to convert IP3 to PP-IP5 (IP7), a key metabolite promoting stress tolerance, metabolic adaptation and fungal dissemination to the brain. We have now directly characterized the enzymatic activity of Arg1, demonstrating that it is a dual specificity (IP3/IP4) kinase producing IP5. We showed previously that IP5 is further phosphorylated by Ipk1 to produce IP6, which is a substrate for the synthesis of PP-IP5 by Kcs1. Phenotypic comparison of the arg1Δ and kcs1Δ deletion mutants (both PP-IP5-deficient) reveals that arg1Δ has the most deleterious phenotype: while PP-IP5 is essential for metabolic and stress adaptation in both mutant strains, PP-IP5 is dispensable for virulence-associated functions such as capsule production, cell wall organization, and normal N-linked mannosylation of the virulence factor, phospholipase B1, as these phenotypes were defective only in arg1Δ. The more deleterious arg1Δ phenotype correlated with a higher rate of arg1Δ phagocytosis by human peripheral blood monocytes and rapid arg1Δ clearance from lung in a mouse model. This observation is in contrast to kcs1Δ, which we previously reported establishes a chronic, confined lung infection. In summary, we show that Arg1 is the most crucial IPK for cryptococcal virulence, conveying PP-IP5-dependent and novel PP-IP5-independent functions.
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Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Criptococosis/microbiología , Cryptococcus neoformans/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Proteínas Bacterianas/genética , Pared Celular/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Modelos Animales de Enfermedad , Femenino , Homeostasis , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Ratones , Ratones Endogámicos BALB C , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , VirulenciaRESUMEN
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.
Asunto(s)
Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Nucleosomas/química , Mapeo de Interacción de Proteínas/métodos , Animales , Artefactos , Western Blotting , Escherichia coli , Células HEK293 , Células HeLa , Histona Desacetilasa 1/química , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Subunidades de Proteína , Conejos , Proteínas Recombinantes de Fusión/química , ReticulocitosRESUMEN
Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).
Asunto(s)
ADN Helicasas/química , Proteínas de Unión al ADN/química , Resonancia Magnética Nuclear Biomolecular , Isótopos de Carbono , Humanos , Isótopos de Nitrógeno , Dominios Proteicos , Estructura Secundaria de Proteína , TritioRESUMEN
The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.
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ADN Helicasas/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.