Integrative structural modeling reveals functional molecular switches of human G protein-coupled bittertaste receptors

On the human tongue, the bitter taste depends on a large family of 25 taste receptors type 2 (TAS2R) belonging to the G protein-coupled receptor (GPCR) family and classified distantly related to class A GPCR. To date, the experimental structures have not been determined for any TAS2R and key residues controlling their function are still under debate. Here we streamline the modeling of these receptors using an integrative approach combining sequence analysis, molecular modeling and site-directed mutagenesis followed by functional assays. We provide a general approach for modeling all mammal TAS2R and identify functional motifs or residues which are central to understand how we perceive bitterness. Above the protocol which is transposable to all TAS2R, the identification of functional molecular switches lays the groundwork for the rational design of chemical modulators of bitter taste receptors. Such ligands will be of broad interest beyond food science since bitter-taste receptors are ectopically expressed in other parts of the human body besides the tongue. Topin et al. Functional molecular switches of mammalian G protein-coupled bitter-taste receptors. Cell. Mol. Life Sci., 2021, 78, 7605-7615. Funding Acknowledgments: This work was supported by the French Ministry of Higher Education and Research [PhD Fellowship], by GIRACT (Geneva, Switzerland) [9th European PhD in Flavor Research Bursaries for first year students] and the Gen Foundation (Registered UK Charity No. 1071026) [a charitable trust which principally provides grants to students/researchers in natural sciences, in particular food sciences/technology]. This work has also been supported by the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR15-IDEX-01. The authors are grateful to the OPAL infrastructure from Universite Cote drAzur and the Universite Cote drAzurrs Center for High-Performance Computing for providing resources and support. FCOI Declarations: Non

Mammalian class I odorant receptors exhibit a conserved vestibular-binding pocket

Odorant receptors represent the largest family of mammalian G protein-coupled receptors. Phylogenetically, they are split into two classes (I and II). By analyzing the entire subclass I odorant receptors sequences, we identified two class I-specific and highly conserved motifs. These are predicted to face each other at the extra-cellular portion of the transmembrane domain, forming a vestibular site at the entrance to the orthosteric-binding cavity. Molecular dynamics simulation combined with site-directed mutagenesis and in vitro functional assays confirm the functional role of this vestibular site in ligand-driven activation. Mutations at this part of the receptor differentially affect the receptor response to four agonists. Since this vestibular site is involved in ligand recognition, it could serve ligand design that targets specifically this sub-genome of mammalian odorant receptors. © 2019, Springer Nature Switzerland AG.1

Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

International audienceG protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed in non-olfactory tissues and emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of a larger mechanism for odor discrimination. Here, we demonstrate that the OR extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid over the orthosteric pocket. We demonstrate using molecular dynamics simulations that ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity, and acts as a gatekeeper of odorant binding. Therefore, we propose the interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. Furthermore, the 3D models created here enabled virtual screening of new OR agonists and antagonists, which exhibited a 70% hit rate in cell assays. Our approach can potentially be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures

p722 ferrocifen loaded lipid nanocapsules improve survival of murine xenografted-melanoma via a potentiation of apoptosis and an activation of CD8+ T lymphocytes

International audienceMetastatic melanoma is a malignant tumor with a poor prognosis. Recent new therapeutics improved the survival of patients at a metastatic stage. However, the low response rate to immunotherapy, explained in part by resistance to apoptosis, needs to develop new strategies. The ferrocifen family represents promising bioorganometallic molecules for melanoma treatment since they show potent anticancer properties. The aim of this study is (i) to evaluate the benefits of a strategy involving encapsulated p722 in lipid nanocapsules (LNC) in B16F10 melanoma mice models and (ii) to compare the beneficial effects with an existing therapy such as anti-CTLA4 mAb. Interestingly, LNC-p722 induces a significant decrease of melanoma cell viability. In vivo data shows a significant improvement in the survival rate and a slower tumor growth with p722-loaded LNC in comparison with anti-CTLA4 mAb. Western blots confirm that LNC-p722 potentiates intrinsic apoptotic pathway. Treatment with LNC-p722 significantly activates CD8+ T lymphocytes compared to treatment with anti-CTLA4 mAb. This study uncovers a new therapeutic strategy with encapsulated p722 to prevent B16F10 melanoma growth and to improve survival of treated mice

Deciphering the Glycan Preference of Bacterial Lectins by Glycan Array and Molecular Docking with Validation by Microcalorimetry and Crystallography

<div><p>Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from <i>Pseudomonas aeruginosa</i> and BambL from <i>Burkholderia ambifaria</i> are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Å resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.</p></div

Flowchart of the docking/analysis protocol.

<p>Flowchart of the docking/analysis protocol.</p

Crystallographic structure of BambL in complex with Le^x tetrasaccharide.

<p>(A) Intramonomeric and (B) intermonomeric sites. Tetrasaccharide and key amino acids are represented as sticks, hydrogen bond to non-fucose carbohydrate residues as dotted lines; 2mF₀-Df_c electron density map contoured at 1σ is shown ad green mesh. (C) Superimposition of Le^x tetrasaccharide from crystal intramonomeric (yellow) and intermonomeric (pink) sites and Le^x trisaccharide from docking (green).</p

Docking of the oligosaccharides in LecB binding site.

<p>a) H type 1, b) H type 2, c) Le^a, d) Le^x, e) sLe^a f) sLe^x and g) A-tri. The docked oligosaccharides are represented as sticks (carbon, oxygen and nitrogen atoms are colored green, red and blue respectively) and the ones from crystal structures are colored orange. Calcium ions are represented as pink spheres. The protein accessible surface is colored in beige for residues comprised within a sphere of 4 Å around the ligand and in blue for residues involved in hydrogen bond with ligand residues (except fucose).</p

Docking of the oligosaccharides in BambL binding site.

<p>a) H type 1, b) H type 2, c) Le^a, d) Le^x, e) sLe^a f) sLe^x and g) A-tri. The color codes are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071149#pone-0071149-g004" target="_blank">Figure 4</a>.</p

Correlations between experimental binding free energies of binding measured from titration microcalorimetry and theoretical ones calculated using MM-GBSA.

<p>Top: LecB, bottom: BambL (bottom). Calculations have been performed with all data (black lines) or with omission of H-2 for LecB or sLea for BambL, respectively.</p