RESUMO
P2X7 receptors are nonselective cation channels that are activated by extracellular ATP and play important roles in inflammation. They differ from other P2X family members by a large intracellular C-terminus that mediates diverse signaling processes that are little understood. A recent cryo-EM study revealed that the C-terminus of the P2X7 receptor forms a unique cytoplasmic ballast domain that possesses a GDP-binding site as well as a dinuclear Zn2+ site. However, the molecular basis for the regulatory function of the ballast domain as well as the interplay between the various ligands remain unclear. Here, we successfully expressed a soluble trimeric P2X7 ballast domain (P2X7BD) and characterized its ligand binding properties using a biophysical approach. We identified calmodulin (CaM)-binding regions within the ballast domain and found that binding of Ca2+-CaM and GDP to P2X7BD have opposite effects on its stability. Small-angle X-ray scattering experiments indicate that Ca2+-CaM binding disrupts the trimeric state of P2X7BD. Our results provide a possible framework for the intracellular regulation of the P2X7 receptor.
Assuntos
Calmodulina , Receptores Purinérgicos P2X7 , Calmodulina/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Ligação Proteica , Sítios de Ligação , Domínios ProteicosRESUMO
Computer-aided drug discovery methods play a major role in the development of therapeutically important small molecules, but their performance needs to be improved. Molecular dynamics simulations in mixed solvents are useful in understanding protein-ligand recognition and improving molecular docking predictions. In this work, we used ethanol as a cosolvent to find relevant interactions for ligands toward protein kinase G, an essential protein of Mycobacterium tuberculosis (Mtb). We validated the hot spots by screening a database of fragment-like compounds and another one of known kinase inhibitors. Next, we performed a pharmacophore-guided docking simulation and found three low micromolar inhibitors, including one with a novel chemical scaffold that we expanded to four derivative compounds. Binding affinities were characterized by intrinsic fluorescence quenching assays, isothermal titration calorimetry, and the analysis of melting curves. The predicted binding mode was confirmed by X-ray crystallography. Finally, the compounds significantly inhibited the viability of Mtb in infected THP-1 macrophages.
Assuntos
Mycobacterium tuberculosis , Sítios de Ligação , Proteínas Quinases Dependentes de GMP Cíclico , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.
RESUMO
During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Sítios de Ligação/genética , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Endocitose/genética , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMO
Membrane proteins (MPs) constitute a large fraction of the proteome, but exhibit physicochemical characteristics that impose challenges for successful sample production crucial for subsequent biophysical studies. In particular, MPs have to be extracted from the membranes in a stable form. Reconstitution into detergent micelles represents the most common procedure in recovering MPs for subsequent analysis. n-dodecyl-ß-D-maltoside (DDM) remains one of the most popular conventional detergents used in production of MPs. Here we characterize the novel DDM analogue 4-trans-(4-trans-propylcyclohexyl)-cyclohexyl α-maltoside (t-PCCαM), possessing a substantially lower critical micelle concentration (CMC) than the parental compound that represents an attractive feature when handling MPs. Using three different types of MPs of human and prokaryotic origin, i.e., a channel, a primary and a secondary active transporter, expressed in yeast and bacterial host systems, respectively, we investigate the performance of t-PCCαM in solubilization and affinity purification together with its capacity to preserve native fold and activity. Strikingly, t-PCCαM displays favorable behavior in extracting and stabilizing the three selected targets. Importantly, t-PCCαM promoted extraction of properly folded protein, enhanced thermostability and provided negatively-stained electron microscopy samples of promising quality. All-in-all, t-PCCαM emerges as competitive surfactant applicable to a broad portfolio of challenging MPs for downstream structure-function analysis.
RESUMO
All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data-analysis platform (https://spc.embl-hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Fluorescência , Mycobacterium tuberculosis/metabolismo , Sistemas On-Line , Temperatura , Termodinâmica , Cinética , Ligantes , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a >or=2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a K(D) of 0.1 muM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/química , Papillomavirus Humano 16/genética , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Estrutura Terciária de Proteína , Integração Viral , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismoRESUMO
Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chemical biologists to synthesize modified histones for use in deciphering the molecular role of methylation in these phenomena, no general method exists to synthesize proteins bearing quantitative site-specific methylation. Here we demonstrate a general method for the quantitative installation of N(epsilon)-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the molecular mechanisms by which histone methylation orchestrates cellular phenomena.
Assuntos
Histonas/genética , Lisina/análogos & derivados , Proteínas Recombinantes/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/biossíntese , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Metilação , Mutagênese Sítio-Dirigida/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.
Assuntos
Detergentes/análise , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana/isolamento & purificação , Detergentes/química , Fluorometria , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Estabilidade Proteica , Solubilidade/efeitos dos fármacosRESUMO
Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.
Assuntos
Proteínas de Ligação a DNA/química , Modelos Moleculares , Proteínas Virais/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Fosforilação , Ácido Poliglutâmico/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Virais/metabolismoRESUMO
In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. However, the precise mechanism of adapter protein assembly at the cell membrane is still unknown. Here, we show that the membrane-proximal domains ENTH of epsin and ANTH of Sla2 form complexes through phosphatidylinositol 4,5-bisphosphate (PIP2) lipid interfaces. Native mass spectrometry reveals how ENTH and ANTH domains form assemblies by sharing PIP2 molecules. Furthermore, crystal structures of epsin Ent2 ENTH domain from S. cerevisiae in complex with PIP2 and Sla2 ANTH domain from C. thermophilum illustrate how allosteric phospholipid binding occurs. A comparison with human ENTH and ANTH domains reveal only the human ENTH domain can form a stable hexameric core in presence of PIP2, which could explain functional differences between fungal and human epsins. We propose a general phospholipid-driven multifaceted assembly mechanism tolerating different adapter protein compositions to induce endocytosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Fúngicas/química , Fosfatidilinositol 4,5-Difosfato/química , Domínios Proteicos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Membrana Celular/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X , Endocitose , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Human syncytial respiratory virus is a nonsegmented negative-strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2-1. The puzzling mechanism of action of M2-1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA-binding activity is located in a monomeric globular `core' domain, a high-resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full-length M2-1 tetramer, with additional electron density for the C-terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two-state equilibrium. These results constitute a further step in the understanding of this unique RNA-binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Ligação a RNA/química , Vírus Sincicial Respiratório Humano/química , Proteínas Virais/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Many chromatin-associated proteins contain two sequence motifs rich in phenylalanine/tyrosine residues of unknown function. These so-called FYRN and FYRC motifs are also found in transforming growth factor beta regulator 1 (TBRG1)/nuclear interactor of ARF and MDM2 (NIAM), a growth inhibitory protein that also plays a role in maintaining chromosomal stability. We have solved the structure of a fragment of TBRG1, which encompasses both of these motifs. The FYRN and FYRC regions each form part of a single folded module (the FYR domain), which adopts a novel alpha + beta fold. Proteins such as the histone H3K4 methyltransferases trithorax and mixed lineage leukemia (MLL), in which the FYRN and FYRC regions are separated by hundreds of amino acids, are expected to contain FYR domains with a large insertion between two of the strands of the beta-sheet.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
A molecular understanding of the biological phenomena orchestrated by lysine N(É)-methylation is impeded by the challenge of producing site-specifically and quantitatively methylated histones. Here, we report a general method that combines genetic code expansion and chemoselective reactions, for the quantitative, site-specific installation of dimethyl-lysine in recombinant histones. We demonstrate the utility of our method by preparing H3K9me2 and show that this modified histone is specifically recognized by heterochromatin protein 1 beta. Extensions of the strategy reported here will allow a range of chemoselective reactions (which have been used for residue-selective, but not site-selective protein modification) to be leveraged for site-specific protein modification.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Histonas/química , Histonas/genética , Imunoprecipitação , Metilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The tumor suppressor p53 can be expressed as different isoforms because of promoter selection and mRNA editing. One isoform, "delta p53" (Delta p53), results from what would be an unusual alternative splicing of exons 7/8 of the p53 gene, conserving the reading frame and generating a novel protein with proposed transcriptional activity essential for the intra S-phase checkpoint. Here, we show that the deletion of the 66 residues that correspond to strand beta10 and the C-terminal helix of the core domain and the interconnecting linker to the tetramerization domain occurring in the Delta p53 isoform leads to a misfolded and unstable protein, prone to form soluble aggregates, which does not bind the p21 promoter site. The complex of coexpressed Delta p53 and flp53 is soluble in vitro and binds poorly to DNA. Our results provide a structural explanation for the dominant-negative effect of Delta p53 and its lack of transcriptional activity.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Linhagem Celular , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Deleção de Sequência , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
The HPV16 E7 oncoprotein is an extended dimer, with a stable and cooperative fold, but that displays properties of "natively unfolded" proteins. Two regions of conserved sequence are found in E7 proteins, where the N-terminus (1-40) includes the retinoblastoma tumor suppressor binding and casein kinase II phosphorylation sites. A fragment containing the highly acidic N-terminal half shows an apparently disordered conformation by far-UV-circular dichroism (CD) at neutral pH, and its hydrodynamic radius is much larger than a neutral peptide of the same length. Trifluoroethanol and micellar concentrations of sodium dodecyl sulfate stabilize a much more helical structure at pH 4.0 than at pH 7.5, while submicellar concentrations of the detergent yield a beta-strand. The shape, pH, and temperature dependence of the CD spectrum at pH 7.5 are indicative of a poly proline type II structure. This structure is stabilized by phosphorylation, which would translate into increased transforming activity in the cell. Thus, the intrinsically disordered properties of the N-terminal module of E7 are responsible for the structural plasticity of the oncoprotein. Although the domain is not a compact and cooperatively folded unit, it is a bona fide functional domain, evolved to maintain a dynamic but extended structure in the cell. These properties allow adaptation to a variety of protein targets and expose the PEST degradation sequence that regulates its turnover in the cell, a modification of which leads to the accumulation of E7 species with consequences in the transformation process.
Assuntos
Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Detergentes/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Proteínas E7 de Papillomavirus , Peptídeos/química , Fosforilação/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The E6 oncoproteins of high-risk HPV types 16 and 18 are involved in the development of cervical cancer. Besides its determinant role in carcinogenic progression, HPV E6 oncoprotein has also been instrumental in elucidating fundamental aspects of p53 function and its ubiquitin-proteasome degradation, with counterpart activities in various DNA tumor viruses. Establishing the conformational state and cellular distribution unequivocally for the endogenous protein in HPV-transformed cell lines derived from carcinomas is essential for understanding the underlying mechanism. Recombinant E6 from high-risk strains 16 and 18 folds into soluble oligomers of approximately 1.2 MDa, which are thermostable and display cooperative loss of tertiary and secondary structure upon chemical denaturation. Antibodies raised against these assemblies locate E6 evenly distributed in the cells. By depleting the polyclonal serum by immunoblocking with monomeric E6, the nuclei of Hela and CaSki cells become completely devoid of label, indicating that monomeric species are mainly localized in the nucleus and that both monomers and oligomers share epitopes. The monomeric species promote degradation of p53 by the proteasome, which correlates with the nuclear localization we describe. In contrast, the oligomeric E6 does not promote p53 degradation, in agreement with its cytoplasmic localization inferred from the immunoneutralization experiments. Our results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Oncogênicas Virais/química , Proteínas Repressoras/química , Sequência de Bases , Linhagem Celular Transformada , Transformação Celular Neoplásica , Transformação Celular Viral , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Papillomavirus Humano 18/fisiologia , Humanos , Modelos Biológicos , Complexos Multiproteicos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/fisiologia , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/virologiaRESUMO
E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/enzimologia , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Conformação Proteica , Desnaturação ProteicaRESUMO
Despite the fact that E7 is a major transforming oncoprotein in papillomavirus, its structure and precise molecular mechanism of action remain puzzling to date. E7 proteins share sequence homology and proteasome targeting properties of tumor suppressors with adenovirus E1A and SV40 T antigen, two other paradigmatic oncoproteins from DNA tumor viruses. High-risk HPV16 E7, a nonglobular dimer with some properties of intrinsically disordered proteins, is capable of undergoing pH-dependent conformational transitions that expose hydrophobic surfaces to the solvent. We found that treatment with a chelating agent produced a protein that can readily assemble into homogeneous spherical particles with an average molecular mass of 790 kDa and a diameter of 50 nm, as determined from dynamic light scattering and electron microscopy. The protein undergoes a substantial conformational transition from coil to beta-sheet structure, with concomitant consolidation of tertiary structure as judged by circular dichroism and fluorescence. The assembly process is very slow, in agreement with a substantial energy barrier caused by structural rearrangements. The resulting particles are highly stable, cooperatively folded, and capable of binding both Congo Red and thioflavin T, reporters of repetitive beta-sheet structures similar to those found in amyloids, although no fibrillar or insoluble material was observed under our experimental conditions.
Assuntos
Proteínas Oncogênicas Virais/química , Papillomaviridae/fisiologia , Montagem de Vírus , Benzotiazóis , Caseína Quinase II , Dicroísmo Circular , Vermelho Congo/química , Dimerização , Corantes Fluorescentes/química , Humanos , Peso Molecular , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/química , Proteínas E7 de Papillomavirus , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Solubilidade , Tiazóis/química , Zinco/químicaRESUMO
High-risk papillomaviruses are known to exert their transforming activity mainly through E7, one of their two oncoproteins. Despite its relevance, no structural information has been obtained that could explain the apparent broad binding specificity of E7. Recombinant E7 from HPV-16 purified to near homogeneity showed two species in gel filtration chromatography, one of these corresponding to a dimer with a molecular weight of 22 kDa, determined by multiangle light scattering. The E7 dimer was isolated for characterization and was shown to undergo a substantial conformational transition when changing from pH 7.0 to 5.0, with an increase in helical structure and increased solvent accessibility to hydrophobic surfaces. The protein was resistant to thermal denaturation even in the presence of SDS, and we show that persistent residual structure in the monomer is responsible for its reported anomalous electrophoretic behavior. The dimer also displays a nonglobular hydrodynamic volume based on gel filtration experiments and becomes more globular in the presence of 0.3 M guanidinium chloride, with hydrophobic surfaces becoming accessible to the solvent, as indicated by the large increase in ANS binding. At low protein concentration, dissociation of the globular E7 dimer was observed, preceding the cooperative unfolding of the structured and extended monomer. Although E7 bears properties that resemble natively unfolded polypeptides, its far-UV circular dichroism spectrum, cooperative unfolding, and exposure of ANS binding sites support a folded and extended, as opposed to disordered and fluctuating, conformation. The large increase in solvent accessibility to hydrophobic surfaces upon small pH decrease within physiological range and in mild denaturant concentrations suggests conformational properties that could have evolved to enable protein-protein recognition of the large number of cellular binding partners reported.