Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.

The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.

Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.

A structural signature motif enlightens the origin and diversification of nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Correlation between equi-partition of aminoacyl-tRNA synthetases and amino-acid biosynthesis pathways.

The partition of aminoacyl-tRNA synthetases (aaRSs) into two classes of equal size and the correlated amino acid distribution is a puzzling still unexplained observation. We propose that the time scale of the amino-acid synthesis, assumed to be proportional to the number of reaction steps (NE) involved in the biosynthesis pathway, is one of the parameters that controlled the timescale of aaRSs appearance. Because all pathways are branched at fructose-6-phosphate on the metabolic pathway, this product is defined as the common origin for the NE comparison. For each amino-acid, the NE value, counted from the origin to the final product, provides a timescale for the pathways to be established. An archeological approach based on NE reveals that aaRSs of the two classes are generated in pair along this timescale. The results support the coevolution theory for the origin of the genetic code with an earlier appearance of class II aaRSs.

Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2ß2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2ß2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2ß2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

Structure of the full human RXR/VDR nuclear receptor heterodimer complex with its DR3 target DNA.

Transcription regulation by steroid hormones and other metabolites is mediated by nuclear receptors (NRs) such as the vitamin D and retinoid X receptors (VDR and RXR). Here, we present the cryo electron microscopy (cryo-EM) structure of the heterodimeric complex of the liganded human RXR and VDR bound to a consensus DNA response element forming a direct repeat (DR3). The cryo-EM map of the 100-kDa complex allows positioning the individual crystal structures of ligand- and DNA-binding domains (LBDs and DBDs). The LBDs are arranged perpendicular to the DNA and are located asymmetrically at the DNA 5'-end of the response element. The structure reveals that the VDR N-terminal A/B domain is located close to the DNA. The hinges of both VDR and RXR are fully visible and hold the complex in an open conformation in which co-regulators can bind. The asymmetric topology of the complex provides the structural basis for RXR being an adaptive partner within NR heterodimers, while the specific helical structure of VDR's hinge connects the 3'-bound DBD with the 5'-bound LBD and thereby serves as a conserved linker of defined length sensitive to mutational deletion.

Architecture of DNA Bound RAR heterodimers.

Nuclear Retinoic Acid receptors (RARs) consist of three subtypes, α, ß, and γ, encoded by separate genes. They function as ligand-dependent transcriptional regulators, forming heterodimers with Retinoid X receptors (RXRs). RARs mediate the effects of retinoic acid (RA), the active metabolite of Vitamin A, and regulate many biological functions such as embryonic development, organogenesis, homeostasis, vision, immune functions, and reproduction. During the two last decades, a number of in-depth structure-function relationship studies have been performed, in particular with drug design perspectives in the therapeutics for cancer, dermatology, metabolic disease, and other human diseases. Recent structural results concerning integral receptors in diverse functional states, obtained using a combination of different methods, allow a better understanding of the mechanisms involved in molecular regulation. The structural data highlight the importance of DNA sequences for binding selectivity and the role of promoter response elements in the spatial organization of the protein domains into functional complexes.

Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors.

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.

Functional insights into the core-TFIIH from a comparative survey.

TFIIH is a eukaryotic complex composed of two subcomplexes, the CAK (Cdk activating kinase) and the core-TFIIH. The core-TFIIH, composed of seven subunits (XPB, XPD, P62, P52, P44, P34, and P8), plays a crucial role in transcription and repair. Here, we performed an extended sequence analysis to establish the accurate phylogenetic distribution of the core-TFIIH in 63 eukaryotic organisms. In spite of the high conservation of the seven subunits at the sequence and genomic levels, the non-enzymatic P8, P34, P52 and P62 are absent from one or a few unicellular species. To gain insight into their respective roles, we undertook a comparative genomic analysis of the whole proteome to identify the gene sets sharing similar presence/absence patterns. While little information was inferred for P8 and P62, our studies confirm the known role of P52 in repair and suggest for the first time the implication of the core TFIIH in mRNA splicing via P34.

Reconstruction of quaternary structure from X-ray scattering by equilibrium mixtures of biological macromolecules.

A recent renaissance in small-angle X-ray scattering (SAXS) made this technique a major tool for the low-resolution structural characterization of biological macromolecules in solution. The major limitation of existing methods for reconstructing 3D models from SAXS is imposed by the requirement of solute monodispersity. We present a novel approach that couples low-resolution 3D SAXS reconstruction with composition analysis of mixtures. The approach is applicable to polydisperse and difficult to purify systems, including weakly associated oligomers and transient complexes. Ab initio shape analysis is possible for symmetric homo-oligomers, whereas rigid body modeling is applied also to dissociating complexes when atomic structures of the individual subunits are available. In both approaches, the sample is considered as an equilibrium mixture of intact complexes/oligomers with their dissociation products or free subunits. The algorithms provide the 3D low-resolution model (for ab initio modeling, also the shape of the monomer) and the volume fractions of the bound and free state(s). The simultaneous fitting of multiple scattering data sets collected under different conditions allows one to restrain the modeling further. The possibilities of the approach are illustrated in simulated and experimental SAXS data from protein oligomers and multisubunit complexes including nucleoproteins. Using this approach, new structural insights are provided in the association behavior and conformations of estrogen-related receptors ERRα and ERRγ. The possibility of 3D modeling from the scattering by mixtures significantly widens the range of applicability of SAXS and opens novel avenues in the analysis of oligomeric mixtures and assembly/dissociation processes.

TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.

Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

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.

Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor.

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3' processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

M-ORBIS: mapping of molecular binding sites and surfaces.

M-ORBIS is a Molecular Cartography approach that performs integrative high-throughput analysis of structural data to localize all types of binding sites and associated partners by homology and to characterize their properties and behaviors in a systemic way. The robustness of our binding site inferences was compared to four curated datasets corresponding to protein heterodimers and homodimers and protein-DNA/RNA assemblies. The Molecular Cartographies of structurally well-detailed proteins shows that 44% of their surfaces interact with non-solvent partners. Residue contact frequencies with water suggest that ∼86% of their surfaces are transiently solvated, whereas only 15% are specifically solvated. Our analysis also reveals the existence of two major binding site families: specific binding sites which can only bind one type of molecule (protein, DNA, RNA, etc.) and polyvalent binding sites that can bind several distinct types of molecule. Specific homodimer binding sites are for instance nearly twice as hydrophobic than previously described and more closely resemble the protein core, while polyvalent binding sites able to form homo and heterodimers more closely resemble the surfaces involved in crystal packing. Similarly, the regions able to bind DNA and to alternatively form homodimers, are more hydrophobic and less polar than previously described DNA binding sites.

Structural insights into the HNF4 biology.

Hepatocyte Nuclear Factor 4 (HNF4) is a transcription factor (TF) belonging to the nuclear receptor (NR) family that is expressed in liver, kidney, intestine and pancreas. It is a master regulator of liver-specific gene expression, in particular those genes involved in lipid transport and glucose metabolism and is crucial for the cellular differentiation during development. Dysregulation of HNF4 is linked to human diseases, such as type I diabetes (MODY1) and hemophilia. Here, we review the structures of the isolated HNF4 DNA binding domain (DBD) and ligand binding domain (LBD) and that of the multidomain receptor and compare them with the structures of other NRs. We will further discuss the biology of the HNF4α receptors from a structural perspective, in particular the effect of pathological mutations and of functionally critical post-translational modifications on the structure-function of the receptor.

In vivo topoisomerase I inhibition attenuates the expression of hypoxia-inducible factor 1α target genes and decreases tumor angiogenesis.

Topoisomerase I is a privileged target for widely used anticancer agents such as irinotecan. Although these drugs are classically considered to be DNA-damaging agents, increasing evidence suggests that they might also influence the tumor environment. This study evaluates in vivo cellular and molecular modifications induced by irinotecan, a topoisomerase I-directed agent, in patient-derived colon tumors subcutaneously implanted in athymic nude mice. Irinotecan was given intraperitoneally at 40 mg/kg five times every 5 d, and expression profiles were evaluated at d 25 in tumors from treated and untreated animals. Unexpectedly, the in vivo antitumor activity of irinotecan was closely linked to a downregulation of hypoxia-inducible factor-1α (HIF1A) target genes along with an inhibition of HIF1A protein accumulation. The consequence was a decrease in tumor angiogenesis leading to tumor size stabilization. These results highlight the molecular basis for the antitumor activity of a widely used anticancer agent, and the method used opens the way for mechanistic studies of the in vivo activity of other anticancer therapies.

Design, synthesis, evaluation, and structure of vitamin D analogues with furan side chains.

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.

Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus.

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.