Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence

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.journal articl

Virtual screening for inhibitors of human aldose reductase

The inhibition of aldose reductase (AR) provides an interesting strategy to prevent the complications of chronic diabetes. Although a large number of different AR inhibitors are known, very few of these compounds exhibit sufficient efficacy in clinical trials. We performed a virtual screening based on the ultrahigh resolution crystal structure of the inhibitor IDD594 in complex with human AR. AR operates on a large scale of structurally different substrates. To achieve this pronounced promiscuity, the enzyme can adapt rather flexibly to its substrates. Likewise, it has a similar adaptability for the binding of inhibitors. We applied a protocol of consecutive hierarchical filters to search the Available Chemicals Directory. In the first selection step, putative ligands were chosen that exhibit functional groups to anchor the anion-binding pocket of AR. Subsequently, a pharmacophore model based on the binding geometry of IDD594 and the mapping of the binding pocket in terms of putative "hot spots" of binding was applied as a second consecutive filter. In a third and final filtering step, the remaining candidate molecules were flexibly docked into the binding pocket of IDD594 with FlexX and ranked according to their estimated DrugScore values. Out of 206 compounds selected by this search and complemented by a cluster analysis and visual inspection, 9 compounds were selected and subjected to biological testing. Of these, 6 compounds showed IC50 values in the micromolar range. According to the proposed binding mode, the two inhibitors BTB02809 (IC50 = 2.4 +/- 0.5 microM) and JFD00882 (IC50 = 4.1 +/- 1.0 microM) both place a nitro group into the hydrophobic specificity pocket of human AR in an orientation coinciding with the position of the bromine atom of IDD594. The interaction of this Br with Thr113 has been identified as a key feature that is responsible for selectivity enhancement

Visualizing the Role of 2’-OH rRNA Methylations in the Human Ribosome Structure

Chemical modifications of RNA have recently gained new attention in biological sciences. They occur notably on messenger RNA (mRNA) and ribosomal RNA (rRNA) and are important for various cellular functions, but their molecular mechanism of action is yet to be understood in detail. Ribosomes are large ribonucleoprotein assemblies, which synthesize proteins in all organisms. Human ribosomes, for example, carry more than 200 modified nucleotides, which are introduced during biogenesis. Chemically modified nucleotides may appear to be only scarcely different from canonical nucleotides, but modifications such as methylations can in fact modulate their chemical and topological properties in the RNA and alter or modulate the overall translation efficiency of the ribosomes resulting in dysfunction of the translation machinery. Recent functional analysis and high-resolution ribosome structures have revealed a large repertoire of modification sites comprising different modification types. In this review, we focus on 2′-O-methylations (2′-O-Me) and discuss the structural insights gained through our recent cryo electron microscopy (cryo-EM) high-resolution structural analysis of the human ribosome, such as their locations and their influence on the secondary and tertiary structures of human rRNAs. The detailed analysis presented here reveals that ribose conformations of the rRNA backbone differ when the 2′-OH hydroxyl position is methylated, with 3′-endo conformations being the default and the 2′-endo conformations being characteristic in that the associated base is flipped-out. We compare currently known 2′-O-Me sites in human rRNAs evaluated using RiboMethSeq and cryo-EM structural analysis and discuss their involvement in several human diseases

Visualization of chemical modifications in the human 80S ribosome structure

Comment in RNA modifications: Ribosomes get decorated. [Nat Chem Biol. 2017]International audienceChemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs

Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm3 crystal

Atomic model building and refinement into high-resolution cryo-EM maps

International audienc

The crystallographic structure of the aldose reductase-IDD552 complex shows direct proton donation from tyrosine 48

The X-ray crystal structure of human aldose reductase (ALR2) in complex with the inhibitor IDD552 was determined using crystals obtained from two crystallization conditions with different pH values (pH 5 and 8). In both structures the charged carboxylic head of the inhibitor binds to the active site, making hydrogen-bond interactions with His110 and Tyr48 and electrostatic interactions with NADP+. There is an important difference between the two structures: the observation of a double conformation of the carboxylic acid moiety of the inhibitor at pH 8, with one water molecule interacting with the main configuration. This is the first time that a water molecule has been observed deep inside the ALR2 active site. Furthermore, in the configuration with the lower occupancy factor the difference electron-density map shows a clear peak (2.5sigma) for the H atom in the hydrogen bond between the inhibitor's carboxylic acid and the Tyr48 side-chain O atom. The position of this peak implies that this H atom is shared between both O atoms, indicating possible direct proton transfer from this residue to the inhibitor. This fact agrees with the model of the catalytic mechanism, in which the proton is donated by the Tyr48 hydroxyl to the substrate. These observations are useful both in drug design and in understanding the ALR2 mechanism

Introduction of Cobalt Ions in γ-Fe 2 O 3 Nanoparticles by Direct Coprecipitation or Postsynthesis Adsorption: Dopant Localization and Magnetic Anisotropy

International audienceThe influence of cobalt doping on the magnetic anisotropy of γ-Fe2O3 nanoparticles has been investigated using two different approaches: (i) simultaneous precipitation of Fe2+, Fe3+, and Co2+ precursors in water and (ii) adsorption of Co2+ ions onto the surface of preformed iron oxide particles followed by diffusion in the solid phase upon heat treatment. The incorporation of small amounts of Co dopants, less than 1 at %, was monitored by magnetization measurements combined with X-ray absorption spectroscopy experiments at the Co K-edge. These latter measurements were carried out in fluorescence mode using a crystal analyzer spectrometer for enhanced sensitivity. Analyses of the X-ray absorption fine structures allowed for unraveling the differences in local atomic structure and valence state of Co in the two series of samples. A thermally activated diffusion in the spinel lattice was observed in the 250−300 °C range, leading to a substantial increase in magnetocrystalline anisotropy. At higher annealing temperature, magnetic anisotropy was still found to increase due to an enhanced surface contribution associated with the dehydroxylation of terminal Fe atoms. This study not only provides direct correlations between magnetic anisotropy and dopant localization in Co-doped γ-Fe2O3 but also demonstrates for the first time that simultaneous coprecipitation of Fe2+, Fe3+, and Co2+ may actually lead to heterogeneous doping, with a significant part of the Co dopants adsorbed at the particle surface

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

International audienceMost nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein-DNA and protein-protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5'-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence