RESUMO
The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4ß,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4ß,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.
Assuntos
Receptores de Calcitriol , Animais , Camundongos , Relação Estrutura-Atividade , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Ratos , Calcitriol/análogos & derivados , Calcitriol/química , Calcitriol/metabolismo , Calcitriol/síntese química , Masculino , Vitamina D/análogos & derivados , Vitamina D/metabolismo , Vitamina D/química , Hipercalcemia/metabolismo , Rim/metabolismoRESUMO
1α,25-dihydroxyvitamin D3 (1,25D3) regulates many physiological processes in vertebrates by binding to the vitamin D receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand-binding domain (LBD) complex from the basal vertebratePetromyzon marinus, sea lamprey (lVDR). Comparison of three-dimensional crystal structures of the lVDR-1,25D3 complex with higher vertebrate VDR-1,25D3 structures suggests that 1,25D3 binds to lVDR similarly to human VDR, but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Furthermore, residue co-evolution analysis of VDR across vertebrates identifies amino acid positions in H9 and the large insertion domain VDR LBD specific as correlated.
Assuntos
Lampreias , Receptores de Calcitriol , Animais , Lampreias/metabolismo , Ligantes , Filogenia , Ligação Proteica , Receptores de Calcitriol/metabolismo , Vitamina DRESUMO
The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.
RESUMO
Efficient optimization of a peptide lead into a drug candidate frequently needs further transformation to augment properties such as bioavailability. Among the different options, foldamers, which are sequence-based oligomers with precise folded conformation, have emerged as a promising technology. We introduce oligourea foldamers to reduce the peptide character of inhibitors of protein-protein interactions (PPI). However, the precise design of such mimics is currently limited by the lack of structural information on how these foldamers adapt to protein surfaces. We report a collection of X-ray structures of peptide-oligourea hybrids in complex with ubiquitin ligase MDM2 and vitaminâ D receptor and show how such hybrid oligomers can be designed to bind with high affinity to protein targets. This work should enable the generation of more effective foldamer-based disruptors of PPIs in the context of peptide lead optimization.
Assuntos
Conformação Proteica em alfa-Hélice/fisiologia , Ureia/química , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.
Assuntos
Sítio Alostérico , Elementos de Resposta , Receptores X de Retinoides/química , Regulação Alostérica , DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores X de Retinoides/metabolismoRESUMO
Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) to regulate gene expression. The heterodimer recognizes the genome via a large and diverse repertoire of DNA response elements. Assessing the binding mode of RAR and RXR with various DNA response elements is important for understanding how they select their binding site and how DNA sequence and topology allosterically regulate RAR function. A number of complementary assays are often employed for analysis of the binding mode. To biochemically and structurally characterize RAR and RXR-DNA complexes, we describe how to express and purify RAR and RXR-DNA binding domains (DBDs) and multidomain constructs. We also describe the use of electrospray ionization mass spectrometry (ESI MS) and isothermal titration calorimetry (ITC) that give information about stoichiometry and binding affinity, as well as our approaches for co-crystallization of RAR and RXR DBDs with DNA.
Assuntos
Proteínas de Ligação a DNA , Receptores do Ácido Retinoico , DNA , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides/genética , TretinoínaRESUMO
Intrinsically disordered proteins (IDPs) constitute a large portion of "Dark Proteome" - difficult to characterize or yet to be discovered protein structures. Here we used conformationally constrained α-methylated amino acids to bias the conformational ensemble in the free unstructured activation domain of transcriptional coactivator ACTR. Different sites and patterns of substitutions were enabled by chemical protein synthesis and led to distinct populations of α-helices. A specific substitution pattern resulted in a substantially higher binding affinity to nuclear coactivator binding domain (NCBD) of CREB-binding protein, a natural binding partner of ACTR. The first X-ray structure of the modified ACTR domain - NCBD complex visualized a unique conformation of ACTR and confirmed that the key α-methylated amino acids are localized within α-helices in the bound state. This study demonstrates a strategy for characterization of individual conformational states of IDPs.
RESUMO
The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.
Assuntos
PPAR gama/genética , Receptor X Retinoide alfa/genética , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Estudos de Coortes , Cristalografia por Raios X , Feminino , Mutação com Ganho de Função , Células HEK293 , Humanos , Masculino , Simulação de Dinâmica Molecular , PPAR gama/química , PPAR gama/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Receptor X Retinoide alfa/metabolismo , Análise de Sequência de DNA , Relação Estrutura-Atividade , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Retinoid X Receptors (RXRs) act as dimer partners for several nuclear receptors including itself, binding to genomic DNA response elements and regulating gene transcription with cell and gene specificity. As homodimers, RXRs bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1) and little variability of this consensus site is observed for natural DR1s. However, these variations are responsible of the modulation of RXR receptors function through differential binding affinity and conformational changes. To further our understanding of the molecular mechanisms underlying RXR-DNA interactions, we examined how RXR DBDs bind to different DR1s using thermodynamics, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. We show that the half-site sequences modulate the binding cooperativity that results from the protein-protein contacts between the two DBDs. Chemical shifts perturbation NMR experiments revealed that sequence variations in half-sites induce changes that propagate from the protein-DNA interface to the dimerization interface throughout the DBD fold.
Assuntos
DNA/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Retinoid X receptors (RXRs) are transcription factors with important functions in embryonic development, metabolic processes, differentiation, and apoptosis. A particular feature of RXRs is their ability to act as obligatory heterodimerization partners of class II nuclear receptors. At the same time, these receptors are also able to form homodimers that bind to direct repeat separated by one nucleotide hormone response elements. Since the discovery of RXRs, most of the studies focused on its ligand binding and DNA binding domains, while its N-terminal domain (NTD) harboring a ligand-independent activation function remained poorly characterized. Here, we investigated the solution properties of the NTD of RXRα alone and in the context of the full-length receptor using small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. We report the solution structure of the full-length homodimeric RXRα on DNA and show that the NTD remains highly flexible within this complex.
Assuntos
Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , DNA/química , DNA/metabolismo , Insetos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/fisiologia , Difração de Raios XRESUMO
Identifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization.
Assuntos
DNA/metabolismo , Espectrometria de Massas/métodos , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Dimerização , Modelos MolecularesRESUMO
Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3' positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements.
Assuntos
Receptores X de Retinoides/genética , Sequência de Bases , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Dimerização , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Elementos de Resposta/genética , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismoRESUMO
Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta , Receptores X de Retinoides/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Células Cultivadas , Imunoprecipitação da Cromatina , Técnicas de Cocultura , Sequência Consenso , Ensaio de Desvio de Mobilidade Eletroforética , Corpos Embrioides/metabolismo , Genoma , Camundongos , Ligação Proteica , Receptores do Ácido Retinoico/química , Sequências Repetitivas de Ácido Nucleico , Receptores X de Retinoides/química , Análise de Sequência de DNA , Titulometria , Transcrição GênicaRESUMO
Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems. Our novel protocol was used to produce in large scale the full-length 160-kDa steroid receptor coactivator 1 (SRC-1), a coregulator of nuclear receptors. The results indicate that we can produce biologically active human SRC-1 in mammalian and insect cells in large scale.
Assuntos
Baculoviridae/genética , Vetores Genéticos/metabolismo , Coativador 1 de Receptor Nuclear/biossíntese , Vaccinia virus/genética , Animais , Linhagem Celular , Cricetinae , Humanos , Coativador 1 de Receptor Nuclear/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Transcription regulation by steroid hormones, vitamin derivatives, and metabolites is mediated by nuclear receptors (NRs), which play an important role in ligand-dependent gene expression and human health. NRs function as homodimers or heterodimers and are involved in a combinatorial, coordinated and sequentially orchestrated exchange between coregulators (corepressors, coactivators). The architecture of DNA-bound functional dimers positions the coregulators proteins. We previously demonstrated that retinoic acid (RAR-RXR) and vitamin D3 receptors (VDR-RXR) heterodimers recruit only one coactivator molecule asymmetrically without steric hindrance for the binding of a second cofactor. We now address the problem of homodimers for which the presence of two identical targets enhances the functional importance of the mode of binding. Using structural and biophysical methods and RAR as a model, we could dissect the molecular mechanism of coactivator recruitment to homodimers. Our study reveals an allosteric mechanism whereby binding of a coactivator promotes formation of nonsymmetrical RAR homodimers with a 21 stoichiometry. Ligand conformation and the cofactor binding site of the unbound receptor are affected through the dimer interface. A similar control mechanism is observed with estrogen receptor (ER) thus validating the negative cooperativity model for an established functional homodimer. Correlation with published data on other NRs confirms the general character of this regulatory pathway.
Assuntos
Núcleo Celular/metabolismo , Sítio Alostérico , Biofísica/métodos , Cristalografia por Raios X/métodos , Dimerização , Humanos , Cinética , Ligantes , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Coativador 1 de Receptor Nuclear/química , Peptídeos/química , Ligação Proteica , Receptores de Calcitriol/química , Receptores do Ácido Retinoico/químicaRESUMO
PPARγ is a key regulator of glucose homeostasis and insulin sensitization. PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks. To understand how coactivators are recognized by the functional heterodimer PPARγ/RXRα and to determine the topological organization of the complexes, we performed a structural study using small angle X-ray scattering of PPARγ/RXRα in complex with DNA from regulated gene and the TIF2 receptor interacting domain (RID). The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.
RESUMO
Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARα bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRα bound to a rexinoid antagonist (LG100754). We observed that RARα exhibits the canonical agonist conformation and RXRα an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a 'phantom ligand' activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the 'phantom ligand effect' of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.
Assuntos
Receptores do Ácido Retinoico/química , Receptores X de Retinoides/química , Retinoides/química , Tetra-Hidronaftalenos/química , Tretinoína/química , Animais , Sítios de Ligação , Ligação Competitiva , Polarização de Fluorescência , Humanos , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Receptor Cross-Talk , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Retinoides/metabolismo , Espalhamento a Baixo Ângulo , Tetra-Hidronaftalenos/metabolismo , Tretinoína/metabolismo , Difração de Raios XRESUMO
A novel cytochrome c(4), the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c(4) has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c(4) in the aerobic respiratory metabolism. The structure of cytochrome c(4) was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c(4) maintained its functional capability at high temperature (60 degrees C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.
Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/ultraestrutura , Modelos Químicos , Oxigênio/química , Thiocapsa roseopersicina/enzimologia , Simulação por Computador , Oxirredução , Conformação Proteica , TemperaturaRESUMO
Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.