Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Are the conformational dynamics of the ghrelin receptor responsible for its signaling selectivity ? – ghredyn

GHREDYN

Are the conformational dynamics of the ghrelin receptor responsible for its functional plasticity ?

Analysis of the conformational dynamics of a G protein coupled receptor and how it relates to its signaling pleiotropy

A current model states that the signaling specificity of G protein-coupled receptors (GPCRs) is determined by the range of degenerate conformational states the receptor explores and how this conformational landscape is modulated by ligands and signaling proteins. In this context, we proposed to explore the relationships between a GPCR, conformational states, the rates and timescale of interconversion between them and the biological output. To this end, we planned to use the ghrelin receptor GHS-R1a. Besides being a prototype for peptide-activated GPCRs, this receptor is a major target for therapeutical applications in the context of the treatment of obesity, addiction, and metabolic diseases such as diabetes. This study is of more than academic interest since stabilization of receptor states is likely the key to modulating ghrelin receptor function in drug design. The data that will emerge from this proposal will be crucial for drug-design to ensure a fine-tuning of ghrelin signaling toward desired therapeutic pathways but away from adverse side effects. In the more general context of GPCR pharmacology, our proposal will advance our current understanding of fundamental basic mechanisms governing GPCR function and will provide us with a toolbox of innovative technologies of general interest for other receptor systems besides the ghrelin one.

It is clear in the GPCR field that only the marriage of solution-state biophysical methods with high-resolution X-ray crystallography will provide a full molecular understanding of receptor functioning. Indeed, crystal structures serve as excellent starting structural points for mapping the ensemble of conformations provided by non-crystallographic methods. We therefore proposed to analyze the local and global changes in ghrelin receptor conformation in response to ligand binding and coupling to signaling partners through an integrated, multidisciplinary strategy that combined innovative tools in biochemistry and chemical synthesis to up-to-date biophysical methods. Specifically, we planned to use fluorescence spectroscopy and solution-state NMR with the purified ghrelin receptor assembled into lipid nanodiscs to get a detailed description of how signaling proteins and ligands from different efficacy and affinity classes control the conformational landscape of this GPCR. Of importance, the methods proposed, in particular solution-state NMR, allow the observation of low-populated states that are invisible with other techniques. In parallel, we planned to combine X-ray crystallography and molecular modeling to assess how these conformational changes pertain to the receptor structure.

As stated above, we planned to analyze ghrelin receptor (GHS-R1a) conformational dynamics using a combination of fluorescence, NMR and crystallography. The main results obtained along these lines are presented below:
Fluorescence – we have applied the unnatural amino-acid technology to the receptor expressed in E. coli to label it with a fluorophore at specific positions. This opened the way to the analysis of GHS-R1a conformational dynamics using time-resolved fluorescence transfer. By doing so, we provide direct evidence that (i) unliganded GHS-R1a exists as in mixture of different conformational states; (ii) ligands from different pharmacological classes affect the relative distribution of these conformations ; (ii) the receptor is pre-coupled to the G protein; (iv) receptor activation results in the rearrangement of the preformed receptor:G protein complex. These data have been published in PNAS this year.
NMR – we have first developed an unprecedented isotope-labeling scheme, i.e. 13C and protonated methyl probes immersed in a homogeneous fully deuterated dipolar environment. We subsequently set-up a protocol to assemble the labeled receptor into lipid discs whose size is compatible with solution-state NMR. By doing so, we obtained NMR spectra of unprecedented resolution in the field.
Crystallization – we have first devised an assay to identify ligands favorable to crystallization among all synthesized in our institute. This fluorescence-based assay allowed us to select a couple of ligands with very low dissociation properties that should be perfectly adapted to crystallization. In parallel, we developed the expression of the purified receptor in both insect and bacterial expression systems. This actually allows us to produce purified GHS-R1a fused to lysozyme T4L in mg amounts. A first series of crystallization trials have been carried out that have allowed us to identify a series of conditions under which microcrystals have been obtained.

Based on the results already obtained we will:
- extend the fluorescence analysis to the influence of lipids and dimerization on receptor conformation;
- take advantage of our original labeling methodology (13C and protonated methyl probes immersed in a homogeneous fully deuterated dipolar environment) that allows us to obtain NMR spectra with unprecedented resolution in the field. We indeed plan to use the very high quality of these NMR data to get a description of the complex conformational landscape the ghrelin receptor can explore and how it can be allosterically modulated by ligands and lipids;
- pursue our crystallization efforts; we have identified conditions where micro-crystal of the ghrelin receptor fused to T4 lysozyme can be obtained. We now plan to optimize these conditions to obtain crystal whose size would be compatible with X-ray diffraction to solve the structure of the receptor.

Damian M, Mary S, Maingot M, M'Kadmi C, Gagne D, Leyris JP, Denoyelle S, Gaibelet G, Gavara L, Garcia de Souza Costa M, Perahia D, Trinquet E, Mouillac B, Galandrin S, Galès C, Fehrentz JA, Floquet N, Martinez J, Marie J, Banères JL. (2015) Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex. Proc Natl Acad Sci U S A. 112:1601-6.

Ghrelin is a peptide hormone that plays a critical role in a variety of physiological processes including endocrine, metabolic, cardiovascular, immunological and reward from both natural (food) and artificial compounds (alcohol, drugs). These physiological effects are all mediated by its membrane receptor, GHS-R1a, which belongs to the G protein-coupled receptor (GPCR) superfamily. As most GPCRs, GHS-R1a is remarkably versatile, being able to activate multiple G protein-dependent and independent signaling pathways. This complex signaling behavior is likely responsible for the diversity in biological effects elicited by ghrelin. Given the multiple effects of ghrelin, its receptor is a prominent pharmacological target in biomedicine with potential clinical applications in various conditions such as management of disease-induced cachexia, obesity, treatment of patients with diabetes, GH deficiency, and drug or alcohol use disorders. However, our ability to fully exploit the therapeutic potential of the ghrelin receptor is actually limited by an incomplete understanding of how signaling efficacy and selectivity are regulated.
A current model states that the signaling specificity of GPCRs is determined by the range of degenerate conformational states the receptor explores and how this conformational landscape is modulated by ligands and signaling proteins. In this context, we propose to explore the relationships between ghrelin receptor conformational states, the rates and timescale of interconversion between them and the biological output. This is of more than academic interest since stabilization of receptor states is likely the key to modulating ghrelin receptor function in drug design. It is clear in the GPCR field that only the marriage of solution-state biophysical methods with high-resolution X-ray crystallography will provide a full molecular understanding of receptor functioning. Indeed, crystal structures serve as excellent starting structural points for mapping the ensemble of conformations provided by non-crystallographic methods. We therefore propose to analyze the local and global changes in ghrelin receptor conformation in response to ligand binding and coupling to signaling partners through an integrated, multidisciplinary strategy that will combine innovative tools in biochemistry and chemical synthesis to up-to-date biophysical methods. Specifically, we will use single-molecule fluorescence spectroscopy (Task 1) and solution-state NMR (Task 2) with the purified ghrelin receptor assembled into lipid nanodiscs to get a detailed description of how signaling proteins and ligands from different efficacy and affinity classes control the conformational landscape of this GPCR. Of importance, the methods proposed, in particular solution-state NMR, will allow the observation of low-populated states that are invisible with other techniques as well as the analysis of the dynamics of the receptor over a wide range of timescales, ranging from pico- to milliseconds. In parallel, we will combine X-ray crystallography and molecular modeling to assess how these conformational changes pertain to the receptor structure (Task 3). By doing so, we should bring a direct experimental demonstration to the model currently proposed for GPCR-mediated signaling where the biological output is directly dictated by the receptor conformational dynamics. The data that will emerge from this proposal will be crucial for drug-design to ensure a fine-tuning of ghrelin signaling toward desired therapeutic pathways but away from adverse side effects. In the more general context of GPCR pharmacology, our proposal will advance our current understanding of fundamental basic mechanisms governing GPCR function and will provide us with a toolbox of innovative technologies of general interest for other receptor systems besides the ghrelin one.

Project coordination

Jean-Louis BANÈRES (Institut des Biomolécules Max Mousseron, UMR 5247 CNRS)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IGF Institut de Génomique Fonctionnelle, UMR 5203-U661
UMR7099 UMR7099 CNRS
CNRS-ICSN Institut de Chimie des Substances Naturelles, UPR 2301
IBMM Institut des Biomolécules Max Mousseron, UMR 5247 CNRS

Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

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