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Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5L observed in the tobacco model.

Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.

The compound [Fe(tvp)$_2$(NCS)$_2$] · CH$_3$OH, where tvp is 1,2-di-(4-pyridyl)-ethylene, has been synthesized and characterized by x-ray single-crystal diffraction. It consists of two perpendicular, two-dimensional networks organized in parallel stacks of sheets made up of edge-shared [Fe(II)]$_4$ rhombuses. The fully interlocked networks define large square channels in the [001] direction. Variable-temperature magnetic susceptibility measurements and Mossbauer studies reveal that this compound shows low-spin to high-spin crossover behavior in the temperature range from 100 to 250 kelvin. The combined structural and magnetic characterization of this kind of compound is fundamental for the interpretation of the mechanism leading to the spin crossover, which is important in the development of electronic devices such as molecular switches.

Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca 2+ activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca 2+ , AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing. Eukaryotic cell plasma membranes possess a mechanism to repair tears caused by stimuli such as mechanical stress. The authors demonstrate that annexin-A5, when assembled into two-dimensional arrays in the presence of calcium, is required for membrane repair.

The first ferritin structure refined at the atomic level has been achieved on recombinant mouse L-chain apoferritin (rMoLF) crystals. These latter diffract to 1.2 Å resolution under cryogenic conditions. When cryo-cooling the sample, the thermal disorder usually observed at room temperature is reduced and the low-temperature structure reveals several details concerning the protein putative active sites and their properties. Within the pores built up by the molecular three-fold symmetry axes, the iron entry route to the ferritin cavity, residues H118, D131 and E134, exhibit alternate conformations associated with the binding of partially hydrated cadmium ions, a metal used as a crystallization agent. At the mineral ferrihydrite nucleation center, the electron density maps evidence the orientation of E57, E60, E61 and E64 glutamate side chains (whereas they were observed highly disordered in previous ferritin structures determined at room temperature) and allow a description of the site taking into account the binding geometry of four Cd2+ ions. Moreover, the side chain of residue K140, lying in the vicinity of the ferrihydrite nucleation center, is shown to interact with residue E61. As previously highlighted, this observation confirms the importance of K140 in the rMoLF sequence, as being responsible for the low level of iron incorporation by mousel L-chain ferritin compared to human L-chain ferritin. Finally, the diffusion of small molecules within the ferritin cavity is illustrated here by the presence of ordered molecules of glycerol used as a cryo-protectant, which bind the inner cavity surface of the protein.

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0 Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.

La confrontation de la vision économique et de la définition juridique de la titrisation montre que les deux disciplines suivent finalement un parcours comparable. Le rappel des préoccupations des spécialistes d’économie et de droit conduisent à constater que la recherche de solution passe par une vision ample des problèmes.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Ethosuximide, the first‐line therapy for childhood absence epilepsy, is currently formulated as a syrup (Zarontin®, Pfizer) with a bitter taste and high sugar content, poorly adapted to children, and a ketogenic diet. The collaborative European FP7 project KIEKIDS aimed at developing an innovative sugar‐free, tasteless formulation convenient for pediatric use. This dual Phase‐I study evaluated two granule formulations based on lipid multiparticulate (LMP) technology. Two panels of 6 healthy adult volunteers underwent a randomized, placebo‐controlled, partly blinded, 3‐way cross‐over trial, comparing ethosuximide granules A or B with placebo granules and syrup at single 10 mg/kg doses. Corresponding plasma pharmacokinetic profiles of ethosuximide were compared, along with palatability, safety, and tolerability. The LMP granule A proved suboptimal due to bitterness and adherence to beaker walls, while the optimized granule B revealed excellent palatability, similar to placebo granules, and low adherence to glass. The relative bioavailability of granules A versus syrup, based on dose‐normalized Cmax and AUC0–∞ was 93.7% [90% CI: 76.3–115.1] and 96.1% [91.0–101.5], respectively. For granules B it was 87.6% [81.6–94.0] and 92.5% [88.5–96.6], respectively, with slightly delayed tmax of 0.75 h [0.5–4.05] compared to syrup 0.5 h [0.3–0.8]. Tolerability visual analog scales revealed a trend for statistically non‐significant improvement versus syrup at peak (30 min) for transient dizziness (both granules), fatigue (granules A), and anxiety (granules B). The innovative ethosuximide granule formulation B achieves a suitable profile for pediatric use, being sugar‐free, tasteless, bioequivalent, and well‐tolerated while enabling precise adjustment to body weight. Time profile of plasma ethosuximide concentrations according to formulation.

Atypical chemokine receptor 1 (ACKR1) is a G protein–coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein–binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1–Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.