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G protein-coupled receptors (GPCRs) play a central role in cellular communication, converting external stimuli into intracellular responses. GPCRs bind a very broad panel of ligands, such as hormones, neurotransmitters, peptides and lipids. Ligand binding triggers a series of receptor conformational rearrangements, enabling the coupling to intracellular partners and the activation of signaling cascades. The major breakthrough in GPCRs structural biology of the past decade has considerably advanced our understanding of GPCR activation. However, structural information cannot fully explain the molecular details of GPCRs pharmacology. Biophysical investigations reveal that GPCRs are very dynamic proteins, capable of exploring a wide range of conformational states. Binding to ligands of various pharmacological classes, as well as intracellular effectors and allosteric modulators, can shift the equilibrium between these states and the kinetic of interconversions among the different conformers. Investigation of GPCR dynamic interplay is therefore important to better understand the complex pharmacology and signaling profile of these receptors.

SummaryG protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor (β2AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα Switch regions and the α5 helix that weaken the G protein-receptor interface. Molecular dynamics (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signaling events.

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Cell membranes represent a complex and variable medium in time and space of lipids and proteins. Their physico-chemical properties are determined by lipid components which can in turn influence the biological function of membranes. Here, we used hydrostatic pressure to study the close dynamic relationships between lipids and membrane proteins. Experiments on the β–barrel OmpX and the α–helical BLT2 G Protein-Coupled Receptor in nanodiscs of different lipid compositions reveal conformational landscapes intimately linked to pressure and lipids. Pressure can modify the conformational landscape of the membrane protein per se, but also increases the gelation of lipids, both being monitored simultaneously at high atomic resolution by NMR. Our study also clearly shows that a membrane protein can modulate, at least locally, the fluidity of the bilayer. The strategy proposed herein opens new perspectives to scrutinize the dynamic interplay between membrane proteins and their surrounding lipids.

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12–HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.

Understanding the signal transduction mechanism mediated by the G Protein-Coupled Receptors (GPCRs) in eukaryote cells represents one of the main issues in modern biology. At the molecular level, various biophysical approaches have provided important insights on the functional plasticity of these complex allosteric machines. In this context, X-ray crystal structures published during the last decade represent a major breakthrough in GPCR structural biology, delivering important information on the activation process of these receptors through the description of the three-dimensional organization of their active and inactive states. In complement to crystals and cryo-electronic microscopy structures, information on the probability of existence of different GPCR conformations and the dynamic barriers separating those structural sub-states is required to better understand GPCR function. Among the panel of techniques available, nuclear magnetic resonance (NMR) spectroscopy represents a powerful tool to characterize both conformational landscapes and dynamics. Here, we will outline the potential of NMR to address such biological questions, and we will illustrate the functional insights that NMR has brought in the field of GPCRs in the recent years.

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions., This work was funded by the Biotechnology and Biological Sciences Research Council (Grants BB/M003604/1, BB/I008675/1, and BB/N015126/1) and the Wellcome Trust (Grants 104632 and 109854/Z/15/Z). The mass spectrometry work was supported by the Centre National de la Recherche Scientifique, INSERM (Grant “DYNAMO,” ANR-11-LABEX-0011-01 to M.C.) and the Région Ile de France for cofunding the Le Service d’Analyse des Médicaments et Métabolites Mass Spectrometry Facility at IPSIT. E.P. is the recipient of an Imperial College London Institute of Chemical Biology Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training studentship.

Conformational dynamics of GPCRs are central to their function but are difficult to explore at the atomic scale. Solution-state NMR has provided the major contribution in that area of study during the past decade, despite nonoptimized labeling schemes due to the use of insect cells and, to a lesser extent, yeast as the main expression hosts. Indeed, the most efficient isotope-labeling scheme ever to address energy landscape issues for large proteins or protein complexes relies on the use of 13CH3 probes immersed in a perdeuterated dipolar environment, which is essentially out of reach of eukaryotic expression systems. In contrast, although its contribution has been underestimated because of technical issues, Escherichia coli is by far the best-adapted host for such labeling. As it is now tightly controlled, we show in this review that bacterial expression can provide an NMR spectral resolution never achieved in the GPCR field.

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on theThermotoga maritimaSecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites usingin vitromutagenesis, native mass spectrometry and biochemical analysis ofEscherichia coliSecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

G protein-coupled receptor (GPCR)-mediated signal transduction has a central role in human physiology and implication in many diseases. Despite the tremendous number of X-ray crystallography structures published in the past decade, the molecular mechanisms of ligand-dependent signaling remain to be completed. In particular, very little information is available concerning the implication of receptor dynamics and conformational changes on GPCR ligand efficiency and coupling. In this context, mapping the conformational landscape of GPCRs, and how it is modulated by the membrane environment and allosteric and signaling partners, is fundamental in order to gain a clear picture of how the signaling mechanism proceeds. Solution-state nuclear magnetic resonance (NMR) is a powerful technique to study GPCR energy landscapes, i.e., conformational ensembles along activation and inactivation pathway, and associated kinetic barriers.

International audience; Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes 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.

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization-ion mobility spectrometry-mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120% D2O. In (1)H-(1)H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6% of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.

Structural biology of membrane proteins is hampered by the difficulty to express and purify them in a large amount. Despite recent progress in biophysical methods that have reduced the need of biological materials, membrane protein production remains a bottleneck in the field and will require further conceptual and technological developments. Among the unique 424 membrane protein structures found in protein databases, about half of them come from proteins produced in Escherichia coli. In this chapter, we have reviewed the existing bacterial expression systems. The T7 RNA polymerase-based expression system accounts for up to 62 % of solved heterologous membrane protein structures. Among the dozen of bacterial hosts available, the mutant hosts C41(DE3) and C43(DE3) have contributed to half of the integral membrane protein structures that were solved after production using the T7 expression system. After a general introduction on this expression system, the protocol section of this chapter provides detailed protocols to select bacterial expression mutant hosts and to optimize culture conditions.