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Functional Modulation of a GPCR Conformational Landscape in a Lipid Bilayer Marina Casiraghi, Marjorie Damian, Ewen Lescop, Elodie Point, Karine Moncoq, Nelly Morellet, Daniel Levy, Jacky Marie, Eric Guittet, Jean-Louis Baneres, and Laurent J Catoire J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04432 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 2016
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Journal of the American Chemical Society
Functional Modulation of a GPCR Conformational Landscape in a Lipid Bilayer Marina Casiraghi,1 Marjorie Damian,2 Ewen Lescop,3 Elodie Point,1 Karine Moncoq,1 Nelly Morellet,3 Daniel Levy,4 Jacky Marie,2 Eric Guittet,3 Jean-Louis Banères,2,* Laurent J. Catoire1,* 1
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, F-75005 Paris, France; 2 Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier-ENSCM, Faculté de Pharmacie, 15 Avenue C. Flahault, F-34093 Montpellier, France; 3Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Sud, 91198 Gif-sur-Yvette, France; 4Institut Curie, Centre de Recherche, 75231 Paris, France; UMR 168, CNRS, 75231 Paris, France; and Université Pierre et Marie Curie, F-75248 Paris, France. ABSTRACT: Mapping the conformational landscape of G protein-coupled receptors (GPCR), and in particular how this landscape is modulated by the membrane environment, is required to get a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state NMR combined with an efficient isotope-labeling scheme. This strategy was applied to a typical G protein-coupled receptor, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Thanks to this, we are able to provide direct evidence that BLT2 explores a complex landscape with the occurrence of four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.
INTRODUCTION Deciphering the mechanism of signal transduction through GPCRs is a major issue in biology and the subject of intense research1. The conformational dynamics of GPCRs is central to their signaling plasticity and allosteric regulation2, but still poorly delineated. In particular, the impact of the membrane environment is still poorly understood, although it has been repeatedly demonstrated that it plays a central role on membrane protein structure and dynamics3. Additional biophysical studies besides existing crystallographic analyses are thus required to complete the description of the conformational space of GPCRs and how it can be modulated by ligands, signaling proteins and membrane composition. In this context, solution-state NMR spectroscopy appears as a promising method as it can provide dynamic pictures of molecules at physiological temperatures at the atomic scale over a timescale of picoseconds to seconds and beyond4,5. So far, NMR studies of GPCR conformational exchange have only been carried out for a very limited number of receptors614 . Most of these studies were nevertheless carried out in detergent solutions. Detergents can affect the stability of isolated receptors15 and impact on their conformational equilibrium through a fast chemical exchange at the surface of the protein16. This also precludes a detailed analysis of the influence of the membrane structure on receptor dynamics. Among all these studies, only one was carried out with a receptor in nanodiscs13 but, in this case, the β2-adrenergic receptor was only partially deuterated, dramatically affecting spectral reso-
lution. Indeed, all the NMR studies relied on either protonated or fluorinated methyl groups within fully protonated proteins612 or partially and inhomogeneously deuterated receptor13. However, the impact of modified residues or fluorinated probes on the protein conformational exchange is difficult to gauge. This can be particularly true near binding sites and/or at sites where conformational changes are likely to occur and where probes are usually located for optimal sensitivity to interaction and/or activation. Finally, well-resolved NMR data can be obtained with protonated 7TM helices proteins provided these proteins are rigid, either naturally17 or because of thermostabilizing mutations14. In all other cases, with unmodified proteins, the NMR signals were very broad due to a residual or fully protonated dipolar environment so that detection of subtle differences in chemical shifts was impossible. This arises because even a residual amount of protons can dramatically affect the relaxation properties of the nuclei under investigation. This is of particular importance for the large, slowtumbling complexes being studied in lipid-reconstituted integral membrane proteins. To address all these issues, we devised an original strategy that associates overexpression in Escherichia coli (E. coli), which allows the design of efficient isotope-labeling schemes dedicated to the study of large protein complexes in solution by NMR, and subsequent reconstitution of the receptor in lipid nanodiscs18. This strategy was applied to a prototypical GPCR, the leukotriene B4 receptor BLT2. Thanks to perdeuteration of the receptor, we refined the current model of GPCR activation
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Figure 1. Pharmacology of BLT2 in nanodiscs. (a) Fluorescence anisotropy-monitored stoichiometric titration of LTB4-568 by BLT2-containing nanodiscs (closed circles) or empty nanodiscs (open circles). (b) Fluorescence anisotropy-monitored doseresponse binding of LTB4-568 to BLT2-containing nanodiscs (closed circles) or empty nanodiscs (open circles). (c) Competitive binding data for inhibition of LTB4-568 binding to BLT2-containing nanodiscs in the presence of leukotriene B4 receptors ligand. (d) Variations of the fluorescence anisotropy signal of LTB4-568 as a function of time at two different temperatures in the presence of BLT2-containing nanodiscs (open and closed circles) or empty nanodiscs (open and closed squares). (e) GTPγS binding to Gαiβγ catalyzed by empty or BLT2-containing nanodiscs in the absence of ligand or in the presence of 1µM LTB4 or LY255283. Data are presented as fluorescence intensities in a given condition (I) normalized to that measured under the same condition in the absence of any particle (I(0)). (f) LTB4 or LY255283 saturation of GTPγS binding to Gαi2β1γ2 in the presence of BLT2 containing nanodiscs (open and closed circles) or empty discs (open and closed squares). Inset: timedependent activation profiles. In all cases (a-f), data are represented as mean ± SD of three representative experiments (** p
