RESUMO
The multi-protein complex WRAD, formed by WDR5, RbBP5, Ash2L and Dpy30, binds to the MLL SET domain to stabilize the catalytically active conformation required for histone H3K4 methylation. In addition, the WRAD complex contributes to the targeting of the activated complex to specific sites on chromatin. RbBP5 is central to MLL catalytic activation, by making critical contacts with the other members of the complex. Interestingly its only major structural domain, a canonical WD40 repeat ß-propeller, is not implicated in this function. Here, we present the structure of the RbBP5 ß-propeller domain revealing a distinct, feature rich surface, dominated by clusters of Arginine residues. Our nuclear magnetic resonance binding data supports the hypothesis that in addition to the role of RbBP5 in catalytic activation, its ß-propeller domain is a platform for the recruitment of the MLL complexes to chromatin targets through its direct interaction with nucleic acids.
Assuntos
Proteínas de Ligação a DNA/química , Metilação , Complexos Multiproteicos/química , Proteínas Nucleares/química , Sítios de Ligação , Catálise , Cromatina/química , Cromatina/genética , Proteínas de Ligação a DNA/genética , Histonas/química , Histonas/genética , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Ligação Proteica/genética , Conformação Proteica , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Repetições WD40/genéticaRESUMO
AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr172). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the ß subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr172 AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr172 AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr172 phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr172, AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, the basal level of Thr172 phosphorylation is very low (â¼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKß, Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172 AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr172 phosphorylation. However, in a cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Compostos de Bifenilo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Pironas/farmacologia , Tiofenos/farmacologiaRESUMO
The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an essential role in early development and hematopoiesis. The biological function of MLL1 is mediated by the histone H3K4 methyltransferase activity of the carboxyl-terminal SET domain. We have determined the crystal structure of the MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3 peptide. This structure indicates that, in order to form a well-ordered active site, a highly variable but essential component of the SET domain must be repositioned. To test this idea, we compared the effect of the addition of MLL complex members on methyltransferase activity and show that both RbBP5 and Ash2L but not Wdr5 stimulate activity. Additionally, we have determined the effect of posttranslational modifications on histone H3 residues downstream and upstream from the target lysine and provide a structural explanation for why H3T3 phosphorylation and H3K9 acetylation regulate activity.
Assuntos
Epigênese Genética/fisiologia , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Acetilação , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Histonas/química , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Proteínas Metiltransferases/metabolismo , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The delivery of site-specific post-translational modifications to histones generates an epigenetic regulatory network that directs fundamental DNA-mediated processes and governs key stages in development. Methylation of histone H4 lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic stability and regulation of replication. We present the structure of the histone H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide substrate and S-adenosyl methionine cofactor. Analysis of the structure reveals that the Suv4-20h2 active site diverges from the canonical SET domain configuration and generates a high degree of both substrate and product specificity. Together with supporting biochemical data comparing Suv4-20h1 and Suv4-20h2, we demonstrate that the Suv4-20 family enzymes take a previously mono-methylated H4K20 substrate and generate an exclusively di-methylated product. We therefore predict that other enzymes are responsible for the tri-methylation of histone H4K20 that marks silenced heterochromatin.
Assuntos
Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/classificação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. RESULTS: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. CONCLUSIONS: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology.
RESUMO
As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain.
Assuntos
Histona-Lisina N-Metiltransferase , Lisina , Arginina/metabolismo , Cristalografia por Raios X , Epigênese Genética , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Lisina/metabolismoRESUMO
The conserved Mixed Lineage Leukaemia (MLL) complex deposits activating methyl marks on histone tails through a methyltransferase (MT) activity. Here we provide in vivo evidence that in addition to methylation, the C. elegans MLL-like complex can remove specific methyl marks linked to repression of transcription. This supports the proposed model in which the MLL complex orchestrates both the deposition and the removal of methyl marks to activate transcription. We have uncovered the MLL-like complex in a large-scale RNAi screen designed to identify attenuators of RAS signalling during vulval development. We have also found that the histone acetyltransferase complex, NuA4/TIP60, cooperates with the C. elegans MLL-like complex in the attenuation of RAS signalling. Critically, we show that both complexes regulate a common novel target and attenuator of RAS signalling, AJM-1 (Apical Junction Molecule-1). Therefore, the C. elegans MLL-like complex cooperates with the NuA4/TIP60 complex to regulate the expression of a novel effector, AJM-1.
Assuntos
Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Histona Acetiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Interferência de RNA , Receptores Proteína Tirosina Quinases/metabolismo , Transcrição Gênica , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication, and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by mixed lineage leukemia protein-1 (MLL1), is responsible for histone H3 lysine 4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multiprotein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WD repeat protein-5 (WDR5), and we mapped the complementary site on its partner retinoblastoma-binding protein-5 (RbBP5). We have characterized this interaction by x-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to mixed lineage leukemia activation, and we combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
Assuntos
Histonas/química , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Proteínas Metiltransferases/química , Proteínas/química , Proteínas de Ligação a Retinoblastoma/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Histonas/genética , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metilação , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Mapeamento de Peptídeos , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismoRESUMO
Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34-318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34-318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34-318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34-318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Vírus da Febre Aftosa/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/fisiologia , Motivos de Aminoácidos , Domínio Catalítico , Escherichia coli/metabolismo , Hidrólise , Cinética , Modelos Biológicos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Viral/metabolismo , Proteínas Virais/químicaRESUMO
Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K (D) of 1.1 +/- 0.2 and 0.47 +/- 0.07 microM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the [DGobsH2O] by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be -154 +/- 2 mV for rop-56H/113H and -87.5 +/- 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Heme/metabolismo , Engenharia de Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Eletroquímica , Heme/química , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/metabolismo , TermodinâmicaRESUMO
Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.
Assuntos
Complexo Repressor Polycomb 2/metabolismo , Precursores de RNA/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Quadruplex G , Histonas/metabolismo , Humanos , Camundongos , Nucleossomos/metabolismo , Ligação Proteica , Precursores de RNA/química , Ativação TranscricionalRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated forms of full-length human AMPK α1ß1γ1.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Cromatografia em Gel/métodos , Cristalografia por Raios X/métodos , Proteínas Quinases Ativadas por AMP/isolamento & purificação , Sítios de Ligação , Cromatografia em Gel/instrumentação , Cristalografia por Raios X/instrumentação , Fosforilação , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Histonas/metabolismo , Lisina/metabolismo , Oxirredutases N-Desmetilantes/química , Montagem e Desmontagem da Cromatina , Epigênese Genética , Histonas/química , Humanos , Histona Desmetilases com o Domínio Jumonji , Lisina/química , Metilação , Oxirredutases N-Desmetilantes/metabolismo , Especificidade por SubstratoRESUMO
Jiao and Liu (Research Articles, 16 October 2015, aac4383) reported the crystal structure of the protein complex polycomb repressive complex 2 from Chaetomium thermophilum This landmark structure has brought invaluable insights into the activation mechanism of this essential methyltransferase. However, the analysis of the x-ray data discussed below suggests that the description of oncogenic H3K27M peptide binding to the active site is incorrect.
Assuntos
Histonas/metabolismo , Complexo Repressor Polycomb 2/química , Chaetomium/metabolismo , Metilação , Complexo Repressor Polycomb 1/metabolismoRESUMO
Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification.
Assuntos
Histonas/química , Lisina/química , Complexo Repressor Polycomb 2/química , Domínios Proteicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Domínio Catalítico , Cristalografia por Raios X , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Modelos Moleculares , Mutação , Oncogenes/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Ligação ProteicaRESUMO
Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 µM, cellular EC50 = 0.032 µM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.
Assuntos
Antineoplásicos/farmacologia , Ensaios Clínicos Fase I como Assunto , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Indóis/farmacologia , Linfoma de Células B/tratamento farmacológico , Piperidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Piperidinas/síntese química , Piperidinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase.