RESUMO
SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Domínio Tudor/efeitos dos fármacosRESUMO
PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.
Assuntos
Metilação de DNA , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Calorimetria , Histonas/química , Humanos , Cinética , Espectrometria de Massas , Especificidade por SubstratoRESUMO
WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/metabolismo , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologiaRESUMO
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.
Assuntos
Cristalização/métodos , Cristalografia/métodos , Peptídeo Hidrolases/química , Proteínas/química , Proteínas/ultraestrutura , Conformação ProteicaRESUMO
The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique "chemical fingerprints" for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural "priming" of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.
Assuntos
Citosol/enzimologia , Sulfotransferases/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Estabilidade Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfoadenosina Fosfossulfato/química , Ligação Proteica , Alinhamento de Sequência , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.
Drugs that are small molecules have the potential to block the individual proteins that drive the spread of cancer, but their design is a challenge. This is because they need to get inside the cell and find their target without binding to other proteins on the way. However, small molecule drugs often have an electric charge, which makes it hard for them to cross the cell membrane. Additionally, most proteins are not completely unique, making it harder for the drugs to find the correct target. CARM1 is a protein that plays a role in the spread of breast cancer cells, and scientists are currently looking for a small molecule that will inhibit its action. The group of enzymes that CARM1 belongs to act by taking a small chemical group, called a methyl group, from a molecule called SAM, and transferring it to proteins that switch genes on and off. In the case of CARM1, this changes cell behavior by turning on genes involved in cell movement. Genetically modifying cells so they will not produce any CARM1 stops the spread of breast cancer cells, but developing a drug with the same effects has proved difficult. Existing drugs that can inhibit CARM1 in a test tube struggle to get inside cells and to distinguish between CARM1 and its related enzymes. Now, Cai et al. have modified and tested a CARM1 inhibitor to address these problems, and find out how these small molecules work. At its core, the inhibitor has a structure very similar to a SAM molecule, so it can fit into the SAM binding pocket of CARM1 and its related enzymes. To stop the inhibitor from binding to other proteins, Cai et al. made small changes to its structure until it only interacted with CARM1.Then, to get the inhibitor inside breast cancer cells, Cai et al. cloaked its charged area with a chemical shield, allowing it to cross the cell membrane. Inside the cell, the chemical shield broke away, allowing the inhibitor to attach to CARM1. Analysis of cells showed that this inhibition only affected the cancer cells most likely to spread. Blocking CARM1 switched off genes involved in cell movement and stopped cancer cells from travelling through 3D gels. This work is a step towards making a drug that can block CARM1 in cancer cells, but there is still further work to be done. The next stages will be to test whether the new inhibitor works in other types of cancer cells, in living animals, and in human patient samples.
Assuntos
Neoplasias da Mama/genética , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigenômica/métodos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Algoritmos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Humanos , Cinética , Células MCF-7 , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.
Assuntos
Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Proteínas Metiltransferases/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Domínio Tudor/fisiologia , Acetilação , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Células HEK293 , Histona-Lisina N-Metiltransferase , Histonas/genética , Humanos , Metilação , Camundongos , Células-Tronco Embrionárias Murinas , Ligação Proteica/fisiologia , Proteínas Metiltransferases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Leucemia/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos SCID , Modelos Moleculares , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The nuclear receptor SET domain-containing family of proteins (NSD1, NSD2, and NSD3) is known to mono- and dimethylate lysine 36 of histone H3 (H3K36). Overexpression and translocation of NSDs have been widely implicated in a variety of diseases including cancers. Although the substrate specificity of NSDs has been a subject of many valuable studies, the activity of these proteins has never been fully characterized in vitro. In this study, we present full kinetic characterization of NSD1, NSD2, and NSD3 and provide robust in vitro assays suitable for screening these proteins in a 384-well format using nucleosome as a substrate. Through monitoring the changes in substrate specificity of a series of NSD constructs and using molecular modeling, we show that a basic post-SET extension common to all three NSDs (corresponding to residues 1209 to 1226 of NSD2) is essential for proper positioning on nucleosome substrates.
Assuntos
Histona-Lisina N-Metiltransferase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares/química , Nucleossomos/química , Proteínas Repressoras/química , Soluções Tampão , Clonagem Molecular , Deleção de Genes , Histona Metiltransferases , Histonas/química , Humanos , Concentração Inibidora 50 , Cinética , Lisina/química , Modelos Moleculares , Mutação , Ligação Proteica , Especificidade por SubstratoRESUMO
PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.
Assuntos
Histona-Lisina N-Metiltransferase/química , Sequência de Aminoácidos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide 'reader' domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.
Assuntos
Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Animais , Metilação de DNA , Histonas/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , SoluçõesRESUMO
The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound 2 (K dis = 7 µM), leading to identification of more potent antagonist 47 (K dis = 0.3 µM).
RESUMO
BACKGROUND: The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. METHODOLOGY/PRINCIPAL FINDINGS: The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. CONCLUSIONS: PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical ß-barrel core, an insertion motif between the second and third ß-strands and a C-terminal α-helix bundle. Both the canonical ß-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Assuntos
Histonas/metabolismo , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , TitulometriaRESUMO
UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.