The Hidden Conformation of Human Histo-blood Group Antigen is a Determinant for Recognition by Pathogen Lectins

International audienceHisto-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavours revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, whilst crystallography and molecular dynamics reveal several higher energy open con-formations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le x openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds

Diversification of EPR signatures in site directed spin labeling using a beta-phosphorylated nitroxide

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a β-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches. This journal is © 2014 the Owner Societies

Interaction between odorants and proteins involved in the perception of smell: the case of odorant-binding proteins probed by molecular modelling and biophysical data

This article is published in Flavour and Fragrance Journal as Part II of Special Issue: 13th Weurman Flavour Research Symposium, Zaragoza, Spain, 27th–30th September 2011, edited by Vicente Ferreira (University of Zaragoza).A joint approach that combines molecular modelling and fluorescence spectroscopy is used to study the affinity of an odorant binding protein towards various odorant molecules. We focus on the capability of molecular modelling to rank odorants according to their affinity with this protein, which is involved in the sense of smell. Although ligand-based approaches are unable to propose an accurate model attending to the strength of the bond with the odorant-binding protein, receptor-based structures considering either static or dynamic structure of the protein perform equally to discriminate between good, medium and low affinity odorants. Such approaches will be useful for further rational design of either odorants or bio-inspired sensors

Structure-based virtual screening of bitter taste receptors

International audienceUnderstanding how chemicals code for a certain type of taste is fundamental for the development of a rational method to create new taste modulators. The identification of these new candidates is important for the food industry and would also be beneficial for the pharmacology industry. In humans, the bitter taste depends on a large family of 25 taste receptors type 2 (TAS2Rs) belonging to the G protein-coupled receptor (GPCR) family. They are classified distantly related to class A GPCR and, to date, the experimental structures have not been determined for any TAS2Rs. Here we present a new structure-based virtual screening strategy to expand the chemical space of bitter taste receptors. Combining molecular modeling (based on a recent general approach for modeling all mammal TAS2Rs1) and in vitro functional assays, we identified new active compounds as activators of human TAS2R10. Such agonists will be of broad interest beyond food science since TAS2Rs are ectopically expressed in other parts of the human body besides the tongue

Tracking ligand-binding effect on protein stability by CD spectroscopy

International audienceThe influence of protein-ligand interactions on protein stability is usually assessed by measurements in the liquid phase. CD spectroscopy appears to be a tool of choice to i) measure the conformation of the protein in different phases and to ii) follow the conformational changes of the protein upon binding.We therefore studied the stability of the rat odorant binding protein 3 (OBP3), its ability to remain functional and a preliminary test of its ligand binding specificity in the dry state. Solid-state spectra were performed on dry thin films prepared by drop casting of initial buffered aqueous solutions of the rat OBP3 onto optically transparent CaF2 windows and subsequently dried under low vacuum.We successfully recorded CD spectra of solid-state apo OBP3 from 280 to 130 nm. A previously unknown positive dichroic band became measurable in the solvent-free state at 175 nm. The reproducibility of the solid-state CD spectrum of apo OBP3 was confirmed by measuring several individually prepared films. We then assessed the time-dependent alteration of the protein in this dry environment. No change in the spectra was observed (storage at constant humidity with binary saturated salt solution), highlighting the stability of the OBP films on a monthly basis.Our results revealed that protein folding is not affected during film formation and remains stable over long-time scales

Trace amine associated receptors (TAARs) response to amines are largely affected by sequences variants

International audienc

Structure-based virtual screening of bitter taste receptors

International audienceUnderstanding how chemicals code for a certain type of taste is fundamental for the development of a rational method to create new taste modulators. The identification of these new candidates is important for the food industry and would also be beneficial for the pharmacology industry. In humans, the bitter taste depends on a large family of 25 taste receptors type 2 (TAS2Rs) belonging to the G protein-coupled receptor (GPCR) family. They are classified distantly related to class A GPCR and, to date, the experimental structures have not been determined for any TAS2Rs. Here we present a new structure-based virtual screening strategy to expand the chemical space of bitter taste receptors. Combining molecular modeling (based on a recent general approach for modeling all mammal TAS2Rs1) and in vitro functional assays, we identified new active compounds as activators of human TAS2R10. Such agonists will be of broad interest beyond food science since TAS2Rs are ectopically expressed in other parts of the human body besides the tongue

Tracking ligand-binding effect on protein stability by CD spectroscopy

International audienceThe influence of protein-ligand interactions on protein stability is usually assessed by measurements in the liquid phase. CD spectroscopy appears to be a tool of choice to i) measure the conformation of the protein in different phases and to ii) follow the conformational changes of the protein upon binding.We therefore studied the stability of the rat odorant binding protein 3 (OBP3), its ability to remain functional and a preliminary test of its ligand binding specificity in the dry state. Solid-state spectra were performed on dry thin films prepared by drop casting of initial buffered aqueous solutions of the rat OBP3 onto optically transparent CaF2 windows and subsequently dried under low vacuum.We successfully recorded CD spectra of solid-state apo OBP3 from 280 to 130 nm. A previously unknown positive dichroic band became measurable in the solvent-free state at 175 nm. The reproducibility of the solid-state CD spectrum of apo OBP3 was confirmed by measuring several individually prepared films. We then assessed the time-dependent alteration of the protein in this dry environment. No change in the spectra was observed (storage at constant humidity with binary saturated salt solution), highlighting the stability of the OBP films on a monthly basis.Our results revealed that protein folding is not affected during film formation and remains stable over long-time scales

Trace amine associated receptors (TAARs) response to amines are largely affected by sequences variants

International audienc

Ligand Binding Properties of Odorant-Binding Protein OBP5 from <i>Mus musculus</i>

Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties