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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.

Inertial measurement units (IMUs) represent a technology that is booming in sports right now. The aim of this study was to evaluate the validity of a new application on the use of these wearable sensors, specifically to evaluate a magnet-based timing system (M-BTS) for timing short-duration sports actions using the magnetometer built into an IMU in different sporting contexts. Forty-eight athletes (22.7 ± 3.3 years, 72.2 ± 10.3 kg, 176.9 ± 8.5 cm) and eight skiers (17.4 ± 0.8 years, 176.4 ± 4.9 cm, 67.7 ± 2.0 kg) performed a 60-m linear sprint running test and a ski slalom, respectively. The M-BTS consisted of placing several magnets along the course in both contexts. The magnetometer built into the IMU detected the peak-shaped magnetic field when passing near the magnets at a certain speed. The time between peaks was calculated. The system was validated with photocells. The 95% error intervals for the total times were less than 0.077 s for the running test and 0.050 s for the ski slalom. With the M-BTS, future studies could select and cut the signals belonging to the other sensors that are integrated in the IMU, such as the accelerometer and the gyroscope.

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRα, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π-bulge in helix H7, which was originally built as a regular α-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1–H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRα LBD.

Small differences in turn cycle structure, invisible to the naked eye, could be decisive in improving descent performance. The aim of this study was to assess the influence of turn cycle structure on the performance of elite alpine skiers using an inertial measurement unit (IMU) in different slalom (SL) course settings. Four SL courses were set: a flat-turned (FT), a steep-turned (ST), a flat-straighter (FS) and a steep-straighter (SS). Five elite alpine skiers (21.2 ± 3.3 years, 180.2 ± 5.6 cm, 72.8 ± 6.6 kg) completed several runs at maximum speed for each SL course. A total of 77 runs were obtained. Fast total times correlate with a longer initiation (INI) time in FT, a shorter steering time out of the turn (STEOUT) in the FT and FS and a shorter total steering time (STEIN+OUT) in the FT and SS courses. The linear mixed model used for the analysis revealed that in the FT-course for each second increase in the INI time, the total time is reduced by 0.45 s, and for every one-second increase in the STEOUT and STEIN+OUT times, the total time increases by 0.48 s and 0.31 s, respectively. Thus, to enhance descent performance, the skier should lengthen the INI time and shorten the STEOUT and STEIN+OUT time. Future studies could use an IMU to detect turn phases and analyze them using the other built-in sensors.

Despite the pronounced polarity of C-F bonds, many fluorinated carbon compounds are hydrophobic: a controversial phenomenon known as "polar hydrophobicity". Here, its underlying microscopic mechanisms are explored by ab initio calculations of fluorinated and hydrogenated diamond (111) surfaces interacting with single water molecules. Gradient- and van, der Waals-corrected density functional theory simulations reveal that "polar hydrophobicity" of the fully fluorinated surfaces is caused by a negligible surface/water electrostatic interaction. The densely packed C-F surface dipoles generate a short-range electric field that decays within the core repulsion zone of the surface and hence vanishes in regions accessible by adsorbates. As a result, water physisorption on fully F-terminated surfaces is weak (adsorption energies E-ad < 0.1 eV) and dominated by van der Waals interactions. Conversely, the near-surface electric field generated by loosely packed dipoles on mixed F/H-terminated surfaces has a considerably longer range, resulting in a stronger water physisorption (E-ad > 0.2 eV) that is dominated by electrostatic interactions. The suppression of electrostatic interactions also holds for perfluorinated molecular carbon compounds, thus explaining the prevalent hydrophobicity of fluorocarbons. In general, densely packed polar terminations do not always lead to short-range electric fields. For example, surfaces with substantial electron density spill-out give rise to electric fields with a much slower decay. However, electronic spill-out is limited in F/H-terminated carbon materials. Therefore, our ab initio results, can be reproduced and rationalized by a simple classical point-charge model. Consequently, classical force fields can be used to study the wetting of F/H-terminated diamond, revealing a pronounced correlation between adsorption energies of single H2O molecules and water contact angles.

A chipless radio-frequency identification (chipless-RFID) and sensing system, where tags are read by proximity (near-field) through a switch, is presented. The tags consist of a set of identical resonant elements (split-ring resonators or SRRs), printed or etched at predefined and equidistant positions, forming a linear chain, each SRR providing a bit of information. The logic state (‘1’ or ‘0’) associated with each resonator depends on whether it is present or not in the predefined position. The reader is an array of power splitters used to feed a set of SRR-loaded transmission lines (in equal number to the number of resonant elements, or bits, of the tag). The feeding (interrogation) signal is a harmonic (single-tone) signal tuned to a frequency in the vicinity of the fundamental resonance of the SRRs. The set of SRR-loaded lines must be designed so that the corresponding SRRs are in perfect alignment with the SRRs of the tag, provided the tag is positioned on top of the reader. Thus, in a reading operation, as long as the tag is very close to the reader, the SRRs of the tag modify (decrease) the transmission coefficient of the corresponding reader line (through electromagnetic coupling between both SRRs), and the amplitude of the output signal is severely reduced. Therefore, the identification (ID) code of the tag is contained in the amplitudes of the output signals of the SRR-loaded lines, which can be inferred sequentially by means of a switching system. Unlike previous chipless-RFID systems based on near-field and sequential bit reading, the tags in the proposed system can be merely positioned on top of the reader, conveniently aligned, without the need to mechanically place them across the reader. Since tag reading is only possible if the tag is very close to the reader, this system can be also used as a proximity sensor with applications such as target identification. The proposed chipless-RFID and sensing approach is validated by reading a designed 4-bit tag. For identification purposes, this system is of special interest in applications where a low number of bits suffice, and tag reading by proximity is acceptable (or even convenient). Applications mostly related to secure paper, particularly involving a limited number of items (e.g., exams, ballots, etc.), in order to provide authenticity and avoid counterfeiting, are envisaged. As a proximity sensor, the system may be of use in detecting and distinguishing different targets in applications such as smart packaging.

Summary The bioactive form of vitamin D [1,25(OH) 2 D 3 ] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR). The 3D structures of the VDR ligand-binding domain with 1,25(OH) 2 D 3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDR gem ) that is unresponsive to 1,25(OH) 2 D 3 , but the activity of which is efficiently induced by the gemini ligands. Moreover, we show that many VDR target genes are repressed by unliganded VDR gem and that mineral ion and bone homeostasis are more impaired in VDR gem mice than in VDR null mice, demonstrating that mutations abolishing VDR ligand binding result in more severe skeletal defects than VDR null mutations. As gemini ligands induce VDR gem transcriptional activity in mice and normalize their serum calcium levels, VDR gem is a powerful tool to further unravel both liganded and unliganded VDR signaling.

Aminoacyl-tRNA synthetases catalyse a key reaction in protein biosynthesis. They match the 20 amino acids to the genetic code by specifically attaching them to their adaptors, transfer ribonucleic acid (tRNA) molecules. The reaction proceeds in two steps: the amino acid is first activated by adenosine triphosphate (ATP) to form aminoacyl adenylate, and then the aminoacyl group is transferred to the terminal ribose of tRNA. This family of enzymes is divided into two classes, based on the similarities in primary structure and architecture of the active site domains; the two architectures are characterized by two modes of binding of ATP, the intermediate aminoacyl adenylate and the acceptor end of tRNA, which result in two regioselectivities of amino acid attachment to the terminal ribose of the tRNA. Aminoacyl-tRNA synthetases are modular enzymes; to the central active site module are attached various domains with diverse functions such as tRNA-binding and amino acid editing. The primary subject of this article are structural and functional aspects of these enzymes. Key Concepts Aminoacylation is a key reaction in protein biosynthesis, as it matches the standard 20 amino acids to the genetic code. Aminoacylation is generally catalysed by aminoacyl-tRNA synthetases, a family of 20 enzymes, one for each standard amino acid and a set of corresponding (cognate isoacceptor) tRNAs. Aminoacylation proceeds in two steps, where the amino acid is first activated by ATP to form aminoacyl adenylate, a mixed anhydride, and then transferred to form an ester bond with the terminal ribose of tRNA. Aminoacyl-tRNA synthetases are divided into two classes, based on the similarities in primary structure (sequence) and architecture of their active site domains. Aminoacyl-tRNA synthetases are modular enzymes; to the central active site module are attached various domains with diverse functions such as tRNA-binding and amino acid editing. Aminoacyl-tRNA synthetases are highly specific enzymes, attaching the correct amino acids to the corresponding tRNAs with high fidelity. Aminoacyl-tRNA synthetases of the two classes generally bind the acceptor arm of tRNA in two ways that constitute mirror images of each other, resulting in two different regioselectivities of amino acid attachment to the tRNA. Keywords: structure and function; aminoacylation; translation; protein synthesis; transfer ribonucleic acid (tRNA)

International audience; 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.

Muscle damage induced by inertial exercise performed on a flywheel device was assessed through the serum evolution of muscle enzymes, interleukin 6, and fiber type-specific sarcomere proteins such as fast myosin (FM) and slow myosin (SM). We hypothesized that a model of muscle damage could be constructed by measuring the evolution of serum concentration of muscle proteins following inertial exercise, according to their molecular weight and the fiber compartment in which they are located. Moreover, by measuring FM and SM, the type of fibers that are affected could be assessed. Serum profiles were registered before and 24, 48, and 144 h after exercise in 10 healthy and recreationally active young men. Creatine kinase (CK) and CK-myocardial band isoenzyme increased in serum early (24 h) and returned to baseline values after 48 h. FM increased in serum late (48 h) and remained elevated 144 h post-exercise. The increase in serum muscle enzymes suggests increased membrane permeability of both fast and slow fibers, and the increase in FM reveals sarcomere disruption as well as increased membrane permeability of fast fibers. Consequently, FM could be adopted as a fiber type-specific biomarker of muscle damage.

L’objectiu d’aquest estudi va ser determinar la importancia de les finalitzacions efectuades en la fase d’atac pels jugadors que ocupen el lloc especific d’extrem en handbol. Prioritariament vam valorar la participacio d’aquests jugadors en funcio del resultat final dels partits i de la classificacio final obtinguda pels equips. La investigacio es va centrar en l’analisi de la totalitat d’encontres d’handbol que els equips nacionals de dotze paisos van disputar en els Jocs Olimpics de Pequin 2008. A partir de la utilitzacio de la metodologia observacional com a metodologia especifica per al desplegament de la investigacio, es va construir un instrument ad hoc per dur a terme l’observacio utilitzant el programa informatic Dartfish TeamPro v. 4.5 per al registre de dades. Aquest programa possibilita tant l’obtencio de dades que poden ser valorades estadisticament com la creacio d’un banc d’imatges utilitzables tant des de l’optica de la investigacio com des de la de l’entrenament. Les conclusions d’aquest treball confirmen la relacio entre una major finalitzacio d’accions d’atac del grup d’extrems i l’obtencio de resultats positius tant des del prisma de les variables victoria-derrota com des de la classificacio final.

Summary 1,25-Dihydroxyvitamin D 3 (1,25D), the hormonal form of vitamin D, and several analogs have failed as monotherapies for cancer because of poor efficacy or acquired resistance. However, 1,25D analogs are amenable to bifunctionalization. Preclinical studies have revealed combinatorial effects of 1,25D analogs and histone deacetylase inhibitors (HDACi). Secosteroidal hybrid molecules combining vitamin D receptor (VDR) agonism with HDACi displayed enhanced efficacy but are laborious to synthesize. Here, we have developed easily assembled, fully integrated, non-secosteroidal VDR agonist/HDACi hybrids. The most promising are full VDR agonists with ∼10-fold lower potency than 1,25D. Structure/function studies revealed that antiproliferative activity against 1,25D-resistant squamous carcinoma cells required VDR agonism and HDACi. Remarkably, modeling and X-ray crystallography reveal non-secosteroidal hybrids bind in the VDR ligand binding domain in the opposite orientation of their secosteroidal counterparts.

Nuclear receptors (NRs) are ligand dependent transcription factors that regulate gene expression. A number of in depth structure–function relationship studies have been performed, in particular with drug design perspectives. Recent structural results concerning integral receptors in diverse functional states, obtained using a combination of different methods, now 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. The solution structures of several heterodimer complexes reveal how the DNA directs the positioning of coactivators. In the case of PPARγ-RXRα the comparison with the crystal structure reveals two different conformational states that illustrate the flexibility of the receptors. The results shed light on the dynamics of the molecular recognition process.

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.

El pdf es la versión de autor.-- et al., An improved synthetic route to 1α,25-dihydroxyvitamin D3 des-side chain analogues 2a and 2b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitaminD receptor (hVDR) complexed to one of these analogues, 20(17→18)-abeo-1α,25-dihydroxy-22-homo-21-norvitamin D3 (2a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25-dihydroxyvitamin D3. Vitamin D3 supplements: The synthesis and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D3 with substituents at C18 are described. Crystallographic analysis revealed that the new side chain introduced at C18 adopts the same orientation as the natural side chain at C17 in the parent molecule 1α,25-dihydroxyvitamin D3. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This study was supported by the Spanish Ministry of Education and Science (Grants SAF2004-01885, SAF2007-60341, and SAF2007-67205), the European Union (MRTN-CT-2005-019496, NucSys), the Xunta de Galicia (Projets INCITE08PXIB-209130PR and ACEUIC-2006/XA050), Consolider, Nanobiomed, Fondo de Investigaciones Sanitarias, Fonds voor Wetenschappelijk Onderzoek (FWO) – Vlaanderen (G.553.06 and G.0587.09), CNRS, INSERM, Universit de Strasbourg, ANR, and the European Commission Structural Proteomics in Europe SPINE2-Complexes (LSHG-CT-2006-031220).

In the nuclear receptor of vitamin D (VDR) histidine 305 participates to the anchoring of the ligand. The VDR H305Q mutation was identified in a patient who exhibited the hereditary vitamin D-resistant rickets (HVDRR). We report the crystal structure of human VDR H305Q-ligand binding domain bound to 1α,25(OH)2D3 solved at 1.8 A resolution. The protein adopts the active conformation of the wild-type liganded VDR. A local conformational flexibility at the mutation site weakens the hydrogen bond between the 25-OH with Gln305, thus explaining the lower affinity of the mutant proteins for calcitriol. The structure provides the basis for a rational approach to the design of more potent ligands for the treatment of HVDRR.

A review of the design and synthesis of structural analogs of the vitamin D hormone recently investigated in our laboratories, and the first report on a new class of vitamin D analogs characterized by an aromatic D-ring, is described. Fil: Eduardo, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Santiago de Compostela; España. Consejo Superior de Investigaciones Científicas; España Fil: Fraga, Ramón. Universidad de Santiago de Compostela; España. Consejo Superior de Investigaciones Científicas; España Fil: Sigüeiro, Rita. Universidad de Santiago de Compostela; España. Consejo Superior de Investigaciones Científicas; España Fil: Marco, Maria. Universidad de Santiago de Compostela; España. Consejo Superior de Investigaciones Científicas; España Fil: Rochel, Natacha. Universite Louis Pasteur; Francia Fil: Moras, Dino. Universite Louis Pasteur; Francia Fil: Mouriño, Antonio. Universidad de Santiago de Compostela; España. Consejo Superior de Investigaciones Científicas; España

Peroxisome proliferator-activated receptor-γ nuclear receptor (PPAR-γ) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-γ. Here we use normal-mode analysis of PPAR-γ to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals.

The vitamin D nuclear receptor is a ligand-dependent transcription factor that controls multiple biological responses such as cell proliferation, immune responses, and bone mineralization. Numerous 1 alpha,25(OH)(2)D(3) analogues, which exhibit low calcemic side effects and/or antitumoral properties, have been synthesized. We recently showed that the synthetic analogue (20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D(3) (2a) acts as a 1 alpha,25(OH)(2)D(3) superagonist and exhibits both antiproliferative and prodifferentiating properties in vitro. Using this information and on the basis of the crystal structures of human VDR ligand binding domain (hVDR LBD) bound to 1 alpha,25(OH)(2)D(3), 2 alpha-methyl-1 alpha,25(OH)(2)D(3), or 2a, we designed a novel analogue, 2 alpha-methyl-(20S,23S)-epoxymethano-1 alpha,25-dihydroxyvitamin D(3) (4a), in order to increase its transactivation potency. Here, we solved the crystal structures of the hVDR LBD in complex with the 4a (C23S) and its epimer 4b (C23R) and determined their correlation with specific biological outcomes.

International audience; C/Si switch: Twofold sila-substitution (C/Si exchange) in the RXR-selective retinoids 4 a (SR11237) and 5 a leads to 4 b (disila-SR11237) and 5 b, respectively. Chemistry and biology of the C/Si pairs are reported.SR11237 (BMS649, 4 a) is a pan-RXR-selective retinoid agonist. Its silicon analogue, disila-SR11237 (4 b; twofold C/Si exchange), was prepared in a multistep synthesis by starting from 1,2-bis(ethynyldimethylsilyl)ethane. In addition, the related C/Si analogues 5 a and 5 b, with an indane (disila-indane) instead of a tetraline (disila-tetraline) skeleton, were synthesized. The C/Si pairs 4 a/4 b and 5 a/5 b were studied for their interaction with retinoid receptors and were demonstrated to be highly potent RXR-selective ("rexinoid") agonists. Interestingly, twofold C/Si exchange in the indane moiety of 5 a resulted in a 10-fold increase in biological activity of the corresponding silicon-containing rexinoid 5 b, possibly resulting from an increased receptor affinity or a divergent allosteric effect on co-regulator-binding surfaces. The crystal structures of the ternary complexes formed by 5 a and 5 b, respectively, with the ligand-binding domain of hRXRalpha and a peptide of the co-activator TIF2/GRIP1 revealed additional interactions of the disila analogue 5 b with the H7 and H11 residues, supporting the first option of increased binding affinity. This is the first demonstration of an increase in binding affinity of a ligand to a nuclear receptor by C/Si replacement, thereby adding this C/Si switch strategy to the repertoire of nuclear receptor ligand design.

This is an article Open Access.-- et al., Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of β-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization. © 2008 Elsevier Ltd. All rights reserved., This work was supported by grants G.0508.05 and G.0553.06 from Fonds voor Wetenschappelijk Onderzoek (FWO), NucSys (EU Marie Curie RTN), SAF2007-60341 from Ministerio de Educación y Ciencia and RTICC RD06/0020/0009 from Ministerio de Sanidad y Consumo of Spain, SPINE (QLG2-CT-220-0098) and SPINE2-complexes (LSHG-CT-2006-031220) from the European Commission and by CNRS, INSERM, ULP.

International audience; Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

SummaryVitamin D nuclear receptor (VDR), a ligand-dependent transcriptional regulator, is an important target for multiple clinical applications, such as osteoporosis and cancer. Since exacerbated increase of calcium serum level is currently associated with VDR ligands action, superagonists with low calcium serum levels have been developed. Based on the crystal structures of human VDR (hVDR) bound to 1α,25-dihydroxyvitamin D3 and superagonists—notably, KH1060—we designed a superagonist ligand. In order to optimize the aliphatic side chain conformation with a subsequent entropy benefit, we incorporated an oxolane ring and generated two stereo diasteromers, AMCR277A and AMCR277B. Only AMCR277A exhibits superagonist activity in vitro, but is as calcemic in vivo as the natural ligand. The crystal structures of the complexes between the ligand binding domain of hVDR and these ligands provide a rational approach to the design of more potent superagonist ligands for potential clinical application.

The journal of biological chemistry 291(28), 14430 - 14446(2016). doi:10.1074/jbc.M116.730382, Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases (aaRSs), with two clearly widespread types of enzymes: a dimeric ($\alpha_2$) species present in some bacteria, archaea, and eukaryotes, and a heterotetrameric form ($\alpha_{2}\beta_2$) 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 a-subunit of the $\alpha_{2}\beta_2$ 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 a-subunit in the complex with an analog of glycyl adenylate at 2.8 A 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 AlaRS, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of $\alpha_{2}\beta_2$ GlyRS, convergent with $\alpha_{2}$ 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., Published by Soc., Bethesda, Md.

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4α (HNF4α). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4α and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4α (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4α as well as decreased endogenous HNF4α protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor α, retinoid X receptor α, and thyroid hormone receptor β) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor α and peroxisome proliferator-activated receptor α. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-α (ERα) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERα in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERα remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERα ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERα monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERα ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

Inborn errors of vitamin B12 (cobalamin, Cbl) metabolism are autosomal recessive disorders and have been classified into nine distinct complementation classes (cblA-cblH and mut). Disorders affecting methylcobalamin metabolism cause megaloblastic anemia, which may be accompanied by leukopenia and thrombocytopenia, and a variety of neurological problems. Disorders affecting adenosylcobalamin cause methylmalonic acidemia and metabolic acidosis. Previous studies have shown that cobalamin binds to two enzymes in humans: methylmalonyl-CoA mutase in mitochondria and methionine synthase in the cytosol. In this study, cobalamin binding patterns were analyzed in crude mitochondrial fractions obtained from both control and patient fibroblasts that had been incubated with [57Co]cyanocobalamin. Crude mitochondrial fractions from control fibroblasts confirmed that the majority of [57Co]Cbl eluted with methylmalonyl-CoA mutase. However, in six of the nine disorders, at least one previously unidentified mitochondrial cobalamin binding protein was observed to bind [57Co]Cbl. The proportion of [57Co]Cbl that binds, is increased compared to controls when a deficiency in either adenosylcobalamin synthesis or utilization prevents binding to methylmalonyl-CoA mutase. Furthermore, unique cobalamin binding profiles emerged demonstrating how known mutations in these patients affect cobalamin binding to as yet unidentified proteins.

In their Opinion article, Rastinejad et al. [1] made a comparative analysis between the structural results on full-length receptors (NRs) in solution and those resulting from three crystal structures. The provocative aspects of many statements and more importantly the incorrect assertions and the illusions that can be generated by the paper have prompted us to present the facts. For a good unbiased review with another perspective see [2].

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.

International audience; The general RNA polymerase II transcription factor TFIIE, which is composed of two subunits, has essential roles in both transcription initiation and promoter escape. Electron microscopy analysis of negatively stained human TFIIE showed a large proportion of alpha/beta heterodimers as well as a small proportion of tetramers. Analytical ultracentrifugation, chemical crosslinking, pulldown experiments and cryo-electron microscopy confirmed that TFIIE is a alpha/beta heterodimer in solution. Three-dimensional envelopes of the alpha/beta particles showed an elongated structure composed of three distinct modules. Finally, a model for the quaternary architecture of the complex is proposed that provides a structural framework to discuss the function of TFIIE in the context of RNA polymerase II transcription initiation.

International audience; Human glycosylated haemoglobin A1C is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA1C were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 A at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 A, alpha = 90.00, beta = 125.40, gamma = 90.00 degrees. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (VM) of 9.70 A3 Da(-1) and a solvent content of 49%.

International audience; NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-angstroms resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11-12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11-12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11-12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.

International audience; Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

The fidelity of aminoacylation of tRNA Thr by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.

The estrogen-related receptor (ERR) gamma behaves as a constitutive activator of transcription. Although no natural ligand is known, ERRgamma is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERRgamma ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERRgamma results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ERalpha LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERRgamma does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERRgamma and ERalpha reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERRgamma relative to ERalpha. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERRgamma and reveal structural differences with ERs that explain why ERRgamma does not bind estradiol or raloxifene and will help to design new selective antagonists.

The nuclear receptor of Vitamin D can be activated by a large number of agonist molecules with a wide spectrum in their stereochemical framework. Up to now most of our structural information related to the protein-ligand complex formation is based on an engineered ligand binding domain (LBD) of the human receptor. We now have extended our database, using a wild-type LBD from zebrafish that confirms the previously reported results and allows to investigate the binding of ligands that induce significant conformational changes at the protein level.

Nuclear receptors (NRs) are ligand-dependent transcription factors that control a large number of physiological events through the regulation of gene transcription. NRs function either as homodimers or as heterodimers with retinoid X receptor/ultraspiracle protein (RXR/USP). A structure-based sequence analysis aimed at discovering the molecular mechanism that controls the dimeric association of the ligand-binding domain reveals two sets of differentially conserved residues, which partition the entire NR superfamily into two classes related to their oligomeric behaviour. Site-directed mutagenesis confirms the functional importance of these residues for the dimerization process and/or transcriptional activity. All homodimers belong to class I, in which the related residues contribute a communication pathway of two salt bridges linking helix 1 on the cofactor-binding site to the dimer interface. A salt bridge involving a differentially conserved arginine residue in loop H8-H9 defines the signature motif of heterodimers. RXR/USP and all Caenorhabditis elegans NRs belong to class I, supporting the hypothesis of an earlier emergence of this class.

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bβ, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bβ and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bβ. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.