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The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.
Assuntos
Alanina/análogos & derivados , Histidina , Quinolonas , Receptores de Antígenos de Linfócitos T , Quinolonas/farmacologia , Monoéster Fosfórico Hidrolases/química , Inibidores EnzimáticosRESUMO
Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.
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DNA , Histona Desacetilases , Fatores de Transcrição MEF2 , Proteínas Repressoras , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/metabolismo , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Humanos , Histona Desacetilases/química , Histona Desacetilases/metabolismoRESUMO
Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.
Assuntos
Condensados Biomoleculares , Orotato Fosforribosiltransferase , Orotidina-5'-Fosfato Descarboxilase , Uridina Monofosfato , Humanos , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Pirimidinas/biossíntese , Uridina , Uridina Monofosfato/metabolismoRESUMO
Cancer is a disease with complex pathological process. Current chemotherapy faces problems such as lack of specificity, cytotoxicity, induction of multi-drug resistance and stem-like cells growth. Nanomaterials are materials in the nanorange 1-100 nm which possess unique optical, magnetic, and electrical properties. Nanomaterials used in cancer therapy can be classified into several main categories. Targeting cancer cells, tumor microenvironment, and immune system, these nanomaterials have been modified for a wide range of cancer therapies to overcome toxicity and lack of specificity, enhance drug capacity as well as bioavailability. Although the number of studies has been increasing, the number of approved nano-drugs has not increased much over the years. To better improve clinical translation, further research is needed for targeted drug delivery by nano-carriers to reduce toxicity, enhance permeability and retention effects, and minimize the shielding effect of protein corona. This review summarizes novel nanomaterials fabricated in research and clinical use, discusses current limitations and obstacles that hinder the translation from research to clinical use, and provides suggestions for more efficient adoption of nanomaterials in cancer therapy.
Assuntos
Nanomedicina , Nanoestruturas/uso terapêutico , Neoplasias/terapia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias/patologia , Microambiente TumoralRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.3000901.].
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BACKGROUND: Of the genes that control mitochondrial biogenesis and function, ERRα emerges as a druggable metabolic target to be exploited for cancer therapy. Of the genes mutated in cancer, TP53 remains the most elusive to target. A clear understanding of how mitochondrial druggable targets can be accessed to exploit the underlying mechanism(s) explaining how p53-deficient tumors promote cell survival remains elusive. METHODS: We performed protein-protein interaction studies to demonstrate that ERRα binds to p53. Moreover, we used gene silencing and pharmacological approaches in tandem with quantitative proteomics analysis by SWATH-MS to investigate the role of the ERRα/p53 complex in mitochondrial biogenesis and function in colon cancer. Finally, we designed in vitro and in vivo studies to investigate the possibility of targeting colon cancers that exhibit defects in p53. RESULTS: Here, we are the first to identify a direct protein-protein interaction between the ligand-binding domain (LBD) of ERRα and the C-terminal domain (CTD) of p53. ERRα binds to p53 regardless of p53 mutational status. Furthermore, we show that the ERRα and p53 complex cooperatively control mitochondrial biogenesis and function. Targeting ERRα creates mitochondrial metabolic stresses, such as production of reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP), leading to a greater cytotoxic effect that is dependent on the presence of p53. Pharmacological inhibition of ERRα impairs the growth of p53-deficient cells and of p53 mutant patient-derived colon xenografts (PDX). CONCLUSIONS: Therefore, our data suggest that by using the status of the p53 protein as a selection criterion, the ERRα/p53 transcriptional axis can be exploited as a metabolic vulnerability.
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The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Endocitose , Endossomos/metabolismo , Feminino , Meiose/fisiologia , Oócitos/metabolismo , Progesterona/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas de Xenopus/fisiologia , Xenopus laevisRESUMO
Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Isoproterenol/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
The Timeless (TIM) and it's interacting partner TIPIN protein complex is well known for its role in replication checkpoints and normal DNA replication processes. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding the expression of the TIM/TIPIN protein complex or its potential role in melanoma. Therefore, we investigated the role of this complex in melanoma. To assess the role of the TIM/TIPIN complex in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was also performed. The TIM/TIPIN complex is frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Our results suggest that the TIM/TIPIN complex plays an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Our studies also provide a beginning structural basis for understanding the assembly of the TIM/TIPIN complex. Further mechanistic investigations are needed to determine the complex's potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma.
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Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.
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Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Mutação , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Nanoparticles are getting a great deal of attention in the rapidly developing field of nanomedicine. For example they can be used as drug delivery systems, for imaging applications, or as carriers for synthetic vaccines. Protein-based nanoparticles offer the advantage of biocompatibility and biodegradability thus avoiding some of the major toxicity concerns with nanoparticle associated approaches. Our group has developed self-assembling peptide/protein nanopartices (SAPNs) that are built up from two coiled-coil oligomerization domains joined by a linker region and used them to design subunit vaccines. For drug delivery approaches the SAPNs need to be as small as possible to avoid strong immune responses that could possibly even lead to anaphylaxis. Here we used a computational and biophysical approach to minimize the size of the SAPNs for their use as drug delivery system. We tested different charge distributions on the pentameric and trimeric coiled-coils in silico with molecular dynamics simulations to down-select an optimal design. This design was then investigated in vitro by biophysical methods and we were able to engineer a minimal SAPN of only 11 nm in diameter. Such minimal-sized SAPNs offer new avenues for a safer development as drug delivery systems or other biomedical applications.
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Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização ProteicaRESUMO
BACKGROUND: Various supra-molecular structures form by self-assembly of proteins in a symmetric fashion. Examples of such structures are viruses, some bacterial micro-compartments and eukaryotic vaults. Peptide/protein-based nanoparticles are emerging in synthetic biology for a variety of biomedical applications, mainly as drug targeting and delivery systems or as vaccines. Our self-assembling peptide nanoparticles (SAPNs) are formed by a single peptide chain that consists of two helical coiled-coil segments connected by a short linker region. One helix is forming a pentameric coiled coil while the other is forming a trimeric coiled coil. RESULTS: Here, we were studying in vitro and in silico the effect of the chain length and of point mutations near the linker region between the pentamer and the trimer on the self-assembly of the SAPNs. 60 identical peptide chains co-assemble to form a spherical nanoparticle displaying icosahedral symmetry. We have stepwise reduced the size of the protein chain to a minimal chain length of 36 amino acids. We first used biochemical and biophysical methods on the longer constructs followed by molecular dynamics simulations to study eleven different smaller peptide constructs. We have identified one peptide that shows the most promising mini-nanoparticle model in silico. CONCLUSIONS: An approach of in silico modeling combined with in vitro testing and verification yielded promising peptide designs: at a minimal chain length of only 36 amino acids they were able to self-assemble into proper nanoparticles. This is important since the production cost increases more than linearly with chain length. Also the size of the nanoparticles is significantly smaller than 20 nm, thus reducing the immunogenicity of the particles, which in turn may allow to use the SAPNs as drug delivery systems without the risk of an anaphylactic shock.
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Nanopartículas/química , Peptídeos/química , Sequência de Aminoácidos , Fenômenos Biofísicos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Nanopartículas/ultraestruturaRESUMO
Nanoscale assemblies are a unique class of materials, which can be synthesized from inorganic, polymeric or biological building blocks. The multitude of applications of this class of materials ranges from solar and electrical to uses in food, cosmetics and medicine. In this review, we initially highlight characteristic features of polymeric nanoscale assemblies as well as those built from biological units (lipids, nucleic acids and proteins). We give special consideration to protein nanoassemblies found in nature such as ferritin protein cages, bacterial microcompartments and vaults found in eukaryotic cells and designed protein nanoassemblies, such as peptide nanofibres and peptide nanotubes. Next, we focus on biomedical applications of these nanoscale assemblies, such as cell targeting, drug delivery, bioimaging and vaccine development. In the vaccine development section, we report in more detail the use of virus-like particles and self-assembling polypeptide nanoparticles as new vaccine delivery platforms.
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Sistemas de Liberação de Medicamentos/métodos , Nanofibras , Nanotecnologia/métodos , Nanotubos de Peptídeos , Vacinas , Animais , HumanosRESUMO
GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.
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DNA/metabolismo , Fatores de Transcrição GATA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Fatores de Transcrição GATA/química , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Dedos de ZincoRESUMO
Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and (19)F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.
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Anilidas/química , Anilidas/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Fatores de Regulação Miogênica/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , DNA/química , Células HeLa , Histona Desacetilases/análise , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição MEF2 , Modelos Moleculares , Fatores de Regulação Miogênica/químicaRESUMO
Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels whose activity can be modulated by endogenous neurotransmitters as well as by synthetic ligands that bind the same or distinct sites from the natural ligand. The subtype of α7 nAChR has been considered as a potenial therapeutic target for Alzheimer's disease, schizophrenia and other neurological and psychiatric disorders. Here we have developed a homology model of α7 nAChR based on two high resolution crystal structures with Brookhaven Protein Data Bank (PDB) codes 2QC1 and 2WN9 for threading on one monomer and then for building a pentamer, respectively. A number of small molecule binding sites are identified using Pocket Finder (J. An, M. Tortov, and R. Abagyan, Molecular & Cellular Proteomics, 4.6, 752-761 (2005)) of Internal Coordinate Mechanics (ICM). Remarkably, these computer-identified sites match perfectly with ordered solvent densities found in the high-resolution crystal structure of α1 nAChR, suggesting that the surface cavities in the α7 nAChR model are likely binding sites of small molecules. A high throughput virtual screening by flexible ligand docking of 5008 small molecule compounds was performed at three potential allosteric modulator (AM) binding sites of α7 nAChR using Molsoft ICM software (R. Abagyan, M. Tortov and D. Kuznetsov, J Comput Chem 15, 488-506, (1994)). Some experimentally verified allosteric modulators of α7 like CCMI comp-6, LY 7082101, 5-HI, TQS, PNU-120596, genistein, and NS-1738 ranked among top 100 compounds, while the rest of the compounds in the list could guide further search for new allosteric modulators.
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Biologia Computacional/métodos , Receptores Nicotínicos/química , Solventes , Interface Usuário-Computador , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Animais , Galinhas , Desenho Assistido por Computador , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Ligação de Hidrogênio , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Receptores Nicotínicos/metabolismo , Alinhamento de Sequência , Bibliotecas de Moléculas Pequenas , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1-MEF2B-DNA complex and the HDAC9 (histone deacetylase 9)-MEF2B-DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.
Assuntos
DNA/metabolismo , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/metabolismo , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Fatores de Transcrição MEF2 , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Recent studies suggest that p53 binds predominantly to consensus sites composed of two decameric half-sites with zero spacing in vivo. Here we report the crystal structure of the p53 core domain bound to a full consensus site as a tetramer at 2.13A resolution. Comparison with previously reported structures of p53 dimer:DNA complexes and a chemically trapped p53 tetramer:DNA complex reveals that DNA binding by the p53 core domain is a cooperative self-assembling process accompanied by structural changes of the p53 dimer and DNA. Each p53 monomer interacts with its two neighboring subunits through two different protein-protein interfaces. The DNA is largely B-form and shows no discernible bend, but the central base-pairs between the two half-sites display a significant slide. The extensive protein-protein and protein-DNA interactions explain the high cooperativity and kinetic stability of p53 binding to contiguous decameric sites and the conservation of such binding-site configuration in vivo.