Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Physiological changes in bilayer thickness induced by cholesterol control GPCR rhodopsin function.

We monitored the effect on function of the G-protein-coupled receptor (GPCR) rhodopsin from small, stepwise changes in bilayer thickness induced by cholesterol. Over a range of phosphatidylcholine bilayers with hydrophobic thickness from ≈21 Å to 38 Å, the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium was monitored with UV-visible spectroscopy while ordering of hydrocarbon chains was probed by 2H-NMR. Addition of cholesterol shifted equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (27 Å) and to MI for thicker bilayers, while small bilayer thickness changes within the range of the protein hydrophobic thickness drastically up- or downregulated MII formation. The cholesterol-induced shifts toward MII for thinner membranes correlated with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR, consistent with continuum elastic modeling. The energetic penalty of adding cholesterol to thick bilayers caused rhodopsin oligomerization and a shift toward MI. In membranes of physiological thickness, changes in bilayer mechanical properties induced by cholesterol potentiated the interplay between bilayer and protein thickness resulting in large swings of the MI-MII equilibrium. In membrane containing cholesterol, elastic deformations near the protein are a dominant energetic contribution to the functional equilibrium of the model GPCR rhodopsin.

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2.

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

Cholesterol as a modulator of cannabinoid receptor CB2 signaling.

Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

How Do Ethanolamine Plasmalogens Contribute to Order and Structure of Neurological Membranes?

Ethanolamine plasmalogen (EtnPLA) is a conical-shaped ether lipid and an essential component of neurological membranes. Low stability against oxidation limits its study in experiments. The concentration of EtnPLA in the bilayer varies depending on cell type and disease progression. Here we report on mixed bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine (C18(Plasm)-18:1PE, PLAPE), an EtnPLA lipid subtype, at mole ratios of 2:1, 1:1, and 1:2. We present X-ray diffuse scattering (XDS) form factors F(qz) from oriented stacks of bilayers, related electron-density profiles, and hydrocarbon chain NMR order parameters. To aid future research on EtnPLA lipids and associated proteins, we have also extended the CHARMM36 all-atom force field to include the PLAPE lipid. The ability of the new force-field parameters to reproduce both X-ray and NMR structural properties of the mixed bilayer is remarkable. Our results indicate a thickening of the bilayer upon incorporation of increasing amounts of PLAPE into mixed bilayers, a reduction of lateral area per molecule, and an increase in lipid tail-ordering. The lateral compressibility modulus (KA) calculated from simulations yielded values for PLAPE similar to POPC.

Probing thermostability of detergent-solubilized CB2 receptor by parallel G protein-activation and ligand-binding assays.

G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [35S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.

Permeability of membranes in the liquid ordered and liquid disordered phases.

The functional significance of ordered nanodomains (or rafts) in cholesterol rich eukaryotic cell membranes has only begun to be explored. This study exploits the correspondence of cellular rafts and liquid ordered (Lo) phases of three-component lipid bilayers to examine permeability. Molecular dynamics simulations of Lo phase dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol show that oxygen and water transit a leaflet through the DOPC and cholesterol rich boundaries of hexagonally packed DPPC microdomains, freely diffuse along the bilayer midplane, and escape the membrane along the boundary regions. Electron paramagnetic resonance experiments provide critical validation: the measured ratio of oxygen concentrations near the midplanes of liquid disordered (Ld) and Lo bilayers of DPPC/DOPC/cholesterol is 1.75 ± 0.35, in very good agreement with 1.3 ± 0.3 obtained from simulation. The results show how cellular rafts can be structurally rigid signaling platforms while remaining nearly as permeable to small molecules as the Ld phase.

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules.

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, ß-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein-lipid interaction in the dysfunction of α-syn.

Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II.

Cannabinoid receptor type II (CB2) is an integral membrane protein with seven transmembrane helices that belongs to the large superfamily of rhodopsin-like G protein-coupled receptors. The CB2 is a part of the endocannabinoid system that plays a vital role in regulation of immune response, inflammation, pain, and other metabolic processes. Information about the structure and function of CB2 in cell membranes is essential for development of specific pharmaceuticals that target CB2 signaling. Methodology for recombinant expression, stable isotope labeling, purification, reconstitution into liposomes, and NMR characterization of functionally active CB2 is presented. Uniformly 13C-, 15N-labeled CB2 protein is expressed by fermentation of Escherichia coli in a medium of defined composition under controlled aeration, pH, and temperature and purified by tandem affinity chromatography. The receptor reconstituted into lipid bilayers is suitable for structural studies by solid-state NMR spectroscopy.

Pore hydration states of KcsA potassium channels in membranes.

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes, but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the transmembrane domain of M2 protein of influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid state NMR. In contrast, the M2 proton channel shows a V-shaped water profile across the membrane, with a narrow constriction at the center, like the hourglass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.

Rhodopsin/lipid hydrophobic matching-rhodopsin oligomerization and function.

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0d27-14:1-PC, 16:0d31-16:1-PC, 18:0d35-18:1-PC, or 20:0d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from (2)H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.

Structural interactions of a voltage sensor toxin with lipid membranes.

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.

The role of membrane curvature elastic stress for function of rhodopsin-like G protein-coupled receptors.

The human genome encodes about 800 different G protein-coupled receptors (GPCR). They are key molecules in signal transduction pathways that transmit signals of a variety of ligands such as hormones and neurotransmitters to the cell interior. Upon ligand binding, the receptors undergo structural transitions that either enhance or inhibit transmission of a specific signal to the cell interior. Here we discuss results which indicate that transmission of such signals can be strongly modulated by the composition of the lipid matrix into which GPCR are imbedded. Experimental results have been obtained on rhodopsin, a prototype GPCR whose structure and function is representative for the great majority of GPCR in humans. The data shed light on the importance of curvature elastic stress in the lipid domain for function of GPCR.

The molecular structure of the liquid-ordered phase of lipid bilayers.

Molecular dynamics simulations reveal substructures within the liquid-ordered phase of lipid bilayers. These substructures, identified in a 10 µs all-atom trajectory of liquid-ordered/liquid-disordered coexistence (L(o)/L(d)) are composed of saturated hydrocarbon chains packed with local hexagonal order and separated by interstitial regions enriched in cholesterol and unsaturated chains. Lipid hydrocarbon chain order parameters calculated from the L(o) phase are in excellent agreement with (2)H NMR measurements; the local hexagonal packing is also consistent with (1)H-MAS NMR spectra of the L(o) phase, NMR diffusion experiments, and small-angle X-ray and neutron scattering. The balance of cholesterol-rich to local hexagonal order is proposed to control the partitioning of membrane components into the L(o) regions. The latter have been frequently associated with formation of so-called rafts, platforms in the plasma membranes of cells that facilitate interaction between components of signaling pathways.

Global fold of human cannabinoid type 2 receptor probed by solid-state 13C-, 15N-MAS NMR and molecular dynamics simulations.

The global fold of human cannabinoid type 2 (CB2 ) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state (13)C- and (15)N magic-angle spinning (MAS) NMR, in combination with chemical-shift prediction from a structural model of the receptor obtained by microsecond-long molecular dynamics (MD) simulations. Uniformly (13)C- and (15)N-labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. (13)C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cß, and C=O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the (13) C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥ 1.5 ppm for carbons and ≥ 5.0 ppm for nitrogens). Simulated two-dimensional (13) Cα(i)-(13)C=O(i) and (13)C=O(i)-(15)NH(i + 1) dipolar-interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid-state MAS NMR.

Simulations of anionic lipid membranes: development of interaction-specific ion parameters and validation using NMR data.

Overbinding of ions to lipid head groups is a potentially serious artifact in simulations of charged lipid bilayers. In this study, the Lennard-Jones radii in the CHARMM force field for interactions of Na(+) and lipid oxygen atoms of carboxyl, phosphate, and ester groups were revised to match osmotic pressure data on sodium acetate and electrophoresis data on palmitoyloleoyl phosphatidylcholine (POPC) vesicles. The new parameters were then validated by successfully reproducing previously published experimental NMR deuterium order parameters for dimyristoyl phosphatidylglycerol (DMPG) and newly obtained values for palmitoyloleoyl phosphatidylserine (POPS). Although the increases in Lennard-Jones diameters are only 0.02-0.12 Å, they are sufficient to reduce Na+ binding, and thereby increase surface areas per lipid by 5-10% compared with the unmodified parameters.