Exploration of the dynamic interplay between lipids and membrane proteins by hydrostatic pressure.

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 ß-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.

Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor.

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.

Allosteric modulation of ghrelin receptor signaling by lipids.

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.

Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism.

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing "molecular bellows" model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.

Structure of the agonist 12-HHT in its BLT2 receptor-bound state.

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.

NMR analysis of GPCR conformational landscapes and dynamics.

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.

Illuminating the Energy Landscape of GPCRs: The Key Contribution of Solution-State NMR Associated with Escherichia coli as an Expression Host.

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.

Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies.

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

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.

Synthesis, characterization and applications of a perdeuterated amphipol.

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.

How amphipols embed membrane proteins: global solvent accessibility and interaction with a flexible protein terminus.

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded ß-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.

The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other solubilizing media.

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.

Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation.

A novel type of nonionic amphipols for handling membrane proteins in detergent-free aqueous solutions has been obtained through free-radical homo-telomerization of an acrylamide-based monomer comprising a C(11) alkyl chain and two glucose moieties, using a thiol as transfer reagent. By controlling the thiol/monomer ratio, the number-average molecular weight of the polymers was varied from 8 to 63 kDa. Homopolymeric nonionic amphipols were found to be highly soluble in water and to self-organize, within a large concentration range, into small, compact particles of ~6 nm diameter with a narrow size distribution, regardless of the molecular weight of the polymer. They proved able to trap and stabilize two test membrane proteins, bacteriorhodopsin from Halobium salinarum and the outer membrane protein X of Escherichia coli, under the form of small and well-defined complexes, whose size, composition, and shape were studied by aqueous size-exclusion chromatography, analytical ultracentrifugation, and small-angle neutron scattering. As shown in a companion paper, nonionic amphipols can be used for membrane protein folding, cell-free synthesis, and solution NMR studies (Bazzacco et al. 2012, Biochemistry, DOI: 10.1021/bi201862v).

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

Sulfonated amphipols: synthesis, properties, and applications.

Amphipols (APols) are amphiphatic polymers that keep membrane proteins (MPs) water-soluble. The best characterized and most widely used APol to date, A8-35, comprises a polyacrylate backbone grafted with octyl- and isopropylamine side chains. The nature of its hydrophilic moieties prevents its use at the slightly acidic pH that is desirable to slow down the rate of amide proton exchange in solution NMR studies. We describe here the synthesis and properties of pH-insensitive APols obtained by replacing isopropyles with taurine. Sulfonated APols (SAPols) can be used to trap MPs in the form of small complexes, to stabilize them, and to keep them water-soluble even at low pH. [(15) N,(1) H]-transverse relaxation-optimized spectroscopy NMR spectra obtained at pH 6.8 of a bacterial outer MP folded in SAPols show that the protein is correctly folded. The spectra have a resolution similar to that achieved with A8-35 and reveal water-exposed amide and indole protons whose resonance peaks are absent at pH 8.0.

Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range.

The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the (1)H-(1)H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.

Structure of a GPCR ligand in its receptor-bound state: leukotriene B4 adopts a highly constrained conformation when associated to human BLT2.

G protein-coupled receptors (GPCRs) are key players in signal recognition and cell communication and are among the most important targets for drug development. Direct structural information on the conformation of GPCR ligands bound to their receptors is scarce. Using a leukotriene receptor, BLT2, expressed under a perdeuterated form in Escherichia coli , purified in milligram amounts, and folded to its native state using amphipols, we have solved, by (1)H NMR, the structure of receptor-bound leukotriene B4 (LTB4). Upon binding, LTB4 adopts a highly constrained seahorse conformation, at variance with the free state, where it explores a wide range of conformations. This structure provides an experimentally determined template of a pro-inflammatory compound for further pharmacological studies. The novel approach used for its determination could prove powerful to investigate ligand binding to GPCRs and membrane proteins in general.

Solution NMR mapping of water-accessible residues in the transmembrane beta-barrel of OmpX.

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane beta-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation.

Heteronuclear dipole-to-dipole cross-relaxation has been applied to exploring intermolecular interactions and intramolecular spatial proximities in a large supramolecular structure comprised of a beta-barrel membrane protein, OmpX, in complex with a polymeric surfactant, amphipol A8-35. The experiments, performed in either the laboratory or the rotating frame, reveal the existence of intermolecular contacts between aromatic amino acids and specific groups of the polymer, in addition to intra-protein dipolar interactions, some of them involving carbonyl carbons. This study opens the perspective of collecting by NMR spectroscopy a new kind of through-space structural information involving aromatic and carbonyl (13)C atoms of large proteins.

NMR study of a membrane protein in detergent-free aqueous solution.

One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.