G Protein-coupled Receptors Dynamics Research Articles

Relevance of G protein-coupled receptor (GPCR) dynamics for receptor activation, signalling bias and allosteric modulation.

G protein-coupled receptors (GPCRs) are one of the major drug targets. In recent years, computational drug design for GPCRs has mainly focused on static structures obtained through X-ray crystallography, cryogenic electron microscopy (cryo-EM) or in silico modelling as a starting point for virtual screening campaigns. However, GPCRs are highly flexible entities with the ability to adopt different conformational states that elicit different physiological responses. Including this knowledge in the drug discovery pipeline can help to tailor novel conformation-specific drugs with an improved therapeutic profile. In this review, we outline our current knowledge about GPCR dynamics that is relevant for receptor activation, signalling bias and allosteric modulation. Ultimately, we highlight new technological implementations such as time-resolved X-ray crystallography and cryo-EM as well as computational algorithms that can contribute to a more comprehensive understanding of receptor dynamics and its relevance for GPCR functionality.

Exploring GPCR conformational dynamics using single-molecule fluorescence

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β2 Adrenergic Receptor at the Single-Molecule Level.

G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environment─lipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer.

Dissecting diffusion and oligomerization dynamics of GPCRs in live cells using single-particle tracking.

Crucial insights about the signaling and trafficking mechanisms of G Protein Coupled Receptors (GPCRs) can be derived from their membrane diffusion properties. Previous studies in our lab (Shivnaraine et al., JACS 2016, Li at al., Biophys. J. 2018) have shown that the M2 muscarinic receptor (M2R) can be purified as oligomers, yet the oligomerization dynamics and the implications for signalling in live cells remain elusive. Here we used single-molecule fluorescence techniques, such as single-particle tracking (SPT) and single-molecule photobleaching (smPB), to characterize the dynamic distribution of M2R in HEK293 cells. To that end, receptors have been co-expressed at their N-terminus either with a green fluorescent protein (eGFP), or with a HaloTag for labelling with HaloTag ligand (HTL) dyes, such as JF549 HTL. For comparison, we applied the same techniques to μ-opioid receptors (MORs) in live U2OS cells. For both cases, measured diffusion maps are spatially and temporally heterogeneous, with receptors transitioning between normal and anomalous diffusion regimes. The analysis of experimental data was reinforced by simulations and by control measurements on monomeric (CD86) and dimeric (CD28) membrane proteins. Intensity traces of immobile, single receptor complexes and of monomeric/dimeric controls in the membrane of fixed cells were analyzed using an in-house smPB code based on a Bayesian algorithm (Garry et al., J. Chem. Phys. 2020). The results confirm the presence, correlated with expression levels, of higher order M2R oligomers in cells, and lay out the foundation for subsequent studies of their functional role.

Femtosecond dynamics of rhodopsin revealed by X-ray laser.

G-protein-coupled receptors of the Rhodopsin family are implicated in transmitting signals across biomembranes and are among the most widespread pharmaceutical targets. For G-protein-coupled receptors (GPCRs) like rhodopsin, an important question is how the rapid local dynamics of the ligand initiate the functional protein transitions. Despite the availability of crystal structures of rhodopsin and cryo-trapped intermediates, it is unclear how the protein dynamics are affected by lattice-packing forces or low temperatures. We hypothesize that the crystalline environment inhibits the full range of conformational motions in the visual activation. Here we show how solution X-ray scattering combined with molecular simulations informs the functional dynamics of membrane proteins. To follow the protein structural changes due to light absorption by retinal, we conducted time-resolved X-ray solution scattering (TR-XSS) studies of detergent-solubilized rhodopsin. Femtosecond solution X-ray scattering using an X-ray free electron laser (XFEL) detected the protein structural change in the absence of a crystal lattice and allowed the first direct observation of cis-trans isomerization of retinal bound to visual rhodopsin as it occurred in real time [1]. We discovered a significant difference-scattering signal within the first few time points immediately following the pump laser triggering event. Two scattering components were found within 10 ps after photoactivation at ambient temperature. The first was consistent with structurally similar photo/bathorhodopsin intermediates, and the second due to changes in solvent dynamics. Interpretation of the TR-XSS profiles using molecular dynamics simulations reveal significant atomic displacements in the β-ionone ring, and the amino acids like Tyr268 previously thought to occur later in the photocycle. Our findings demonstrate how X-ray scattering with an XFEL informs the functional dynamics of GPCRs, without artifacts caused by crystal packing or cryo-trapping photointermediates. [1] M.F. Brown et al. (2022) Biophys. J. 121, 193a.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

Cholesterol-Dependent Dynamics of the Serotonin1A Receptor Utilizing Single Particle Tracking: Analysis of Diffusion Modes.

G protein-coupled receptors (GPCRs) are signaling hubs in cell membranes that regulate a wide range of physiological processes and are popular drug targets. Serotonin1A receptors are important members of the GPCR family and are implicated in neuropsychiatric disorders. Cholesterol is a key constituent of higher eukaryotic membranes and is believed to contribute to the segregated distribution of membrane constituents into domains. To explore the role of cholesterol in lateral dynamics of GPCRs, we utilized single particle tracking (SPT) to monitor diffusion of serotonin1A receptors under acute and chronic cholesterol-depleted conditions. Our results show that the short-term diffusion coefficient of the receptor decreases upon cholesterol depletion, irrespective of the method of cholesterol depletion. Analysis of SPT trajectories revealed that relative populations of receptors undergoing various modes of diffusion change upon cholesterol depletion. Notably, in cholesterol-depleted cells, we observed an increase in the confined population of the receptor accompanied by a reduction in diffusion coefficient for chronic cholesterol depletion. These results are supported by our recent work and present observations that show polymerization of G-actin in response to chronic cholesterol depletion. Taken together, our results bring out the interdependence of cholesterol and actin cytoskeleton in regulating diffusion of GPCRs in membranes.

Conformational dynamics in GPCR signaling by NMR

G-protein-coupled receptors (GPCRs) mediate a wide range of cellular responses to various ligands or stimuli, and are the most important drug targets associated with human diseases. While major advances in GPCR structural biology have greatly deepened our understanding of its activation mechanism, the highly complex changes in the structural dynamics of GPCRs during activation remain underdetermined and their links to physiological functions largely unknown. Solution nuclear magnetic resonance (NMR) spectroscopy is an essential technique that allows the characterization of protein structural dynamics at atomic level, and has been applied in the studies of GPCR structural-function relationship in the past decade. Herein, we summarize a few specific studies in which solution NMR methods were employed and provided novel insights into questions difficult to be addressed by other methods.

Modulation of GPCR Rhodopsin Function by Membrane Lipids and Water

G protein‐coupled receptors (GPCRs) are responsible for the transduction of signals across lipid membranes and are the largest family of targets (~35%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. Consequently, understanding the modulation of conformational dynamics of GPCRs by soft matter such as membrane lipids and cellular water in the receptor’s environment is critical to understanding GPCR efficacy and selectivity. Here we show how membrane lipids and cellular water play crucial roles in shaping the energy landscape of GPCR activation, thus acting as allosteric modulators. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native and POPC recombinant membranes after light activation. Using different polyethylene glycol (PEG) solutions, the osmotic pressure on rhodopsin was varied, while shifting of the metarhodopsin equilibrium was probed using UV‐Visible spectroscopy. Our results show a flood of ~80‐100 water molecules into the rhodopsin interior during photoactivation, forming a solvent‐swollen Meta‐II active state. Dehydrating conditions favor Meta‐I through the efflux of water from the protein interior yet simultaneously increase bilayer thickness, which should favor Meta‐II. Because the osmotic effect on the protein is more significant than the effect of the lipid bilayer, the overall equilibrium is generally shifted to Meta‐I. However, small osmolytes favored the Meta‐II state because they can penetrate the protein core, decreasing the osmotic effect on the protein. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta‐I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C‐terminal peptide‐binding isotherms revealed that the G‐prottein binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta‐I state. By contrast, the native lipid membrane environment possesses a negative spontaneous curvature that favors the more expanded state of Meta‐II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment.

Function-related dynamics of GPCRs

G protein-coupled receptors (GPCRs) include various neurotransmitters and hormones, and over 30% of modern drugs target GPCRs. The number of GPCR crystal structures has rapidly increased, and many structures of GPCRs in complexes with their binding partners are being solved by cryo-electron microscopy. However, crystallographic or cryo-electron microscopy data alone cannot fully explain the important features of GPCR signaling determined experimentally. Recent studies have suggested that GPCRs are structurally dynamic, and exchange between multiple conformations. In this respect, NMR methods provide information about the dynamics of proteins over a wide range of frequencies, in aqueous solutions at nearphysiological temperatures. Although NMR studies of GPCRs are challenging due to their innate instability and relatively large molecular weights, recent methodological advances have enabled us to observe the NMR signals of various GPCRs. These NMR studies revealed that GPCRs exist in function-related equilibria between locally different conformations that are simultaneously populated. Here we will describe solution NMR studies that have clarified the function-related conformational dynamics of two GPCRs, β2 adrenergic receptor and adenosine A2A receptor.

Hydration Drives Activation of the G‐Protein‐Coupled Receptor Rhodopsin

G-protein-coupled receptors (GPCRs) are a pivotal superfamily of seven-helix transmembrane receptors responsible for the transduction of stimuli across cellular membranes. They are the targets of ~30% of the pharmaceuticals currently in the market. Understanding how the soft membrane matter (lipids and water) influences the structural changes during the activation of GPCRs is crucial to discovering and developing novel GPCR-targeted drugs [1]. Here, we assessed the effects of hydration on the conformational dynamics of GPCRs by subjecting the archetypical GPCR-rhodopsin in native retinal disk membranes to varying osmotic pressures using polyethylene glycol (PEG) solutions of different molar mass [2,3]. The metastable activation equilibrium of rhodopsin (Keq) after photoexcitation was quantified using UV-visible spectroscopy. Analysis of the results using thermodynamic relations revealed for the first time an influx of ~80-100 water molecules flooding into the rhodopsin interior during photoactivation, validating previous molecular dynamics (MD) simulations [4]. Both applied osmotic pressure and the osmolyte size affected the metarhodopsin equilibrium. Dehydrating conditions generally favored the closed inactive Meta-I state through an efflux of water from the protein interior, while small penetrating osmolytes stabilized the expanded active Meta-II conformation in low concentration regimes. The pH titration curves for the metarhodopsin equilibrium in the presence of various PEG osmolytes showed that hydration is coupled to activation via modulation of the pKA of Glu134 of the conserved E(D)RY sequence motif. By changing water availability, osmotic stress perturbs the dielectric constant characterizing the Glu134 microenvironment and the thermodynamics of its protonation. Activation of rhodopsin is contingent upon the change in electrostatic microenvironments by rearranging water molecules within the protein conformation, dramatically recasting the role of soft matter in biological signal transduction. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] U. Chawla et al. (2020) Angew. Chem. Int. Ed. doi:10.1002/anie.202003342. [3] N. Weerasinghe et al. (2018) Biophys. J. 114, 274a. [4] N. Leioatts et al. (2014) Biochemistry 53, 376−385.

The role of structural dynamics in GPCR-mediated signaling.

G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.

Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse.

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

Dynamic G Protein-Coupled Receptor Signaling Probed by Solution NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for investigating various dynamic features of G protein-coupled receptor (GPCR) signaling. In this review, we focus on NMR techniques to characterize ligand-dependent conformational dynamics of GPCRs as well as the interaction of GPCRs with its environment and ligands. We also describe circumstances under which each technique should be applied, their advantages and disadvantages, and how they can be combined with other strategies to deepen the understanding of GPCR signaling at the molecular level.

Statin-Induced Chronic Cholesterol Depletion Switches GPCR Endocytosis and Trafficking: Insights from the Serotonin1A Receptor.

Endocytosis is a key regulatory mechanism adopted by G protein-coupled receptors (GPCRs) to modulate downstream signaling responses within a stringent spatiotemporal regime. Although the role of membrane lipids has been extensively studied in the context of the function, organization, and dynamics of GPCRs, their role in receptor endocytosis remains largely unexplored. Cholesterol, the predominant sterol in higher eukaryotes, plays a crucial role in maintaining the structure and organization of cell membranes and is involved in essential cellular processes in health and disease. The serotonin1A receptor is a representative GPCR involved in neuronal development and in neuropsychiatric disorders such as anxiety and depression. We recently combined quantitative flow cytometric and confocal microscopic approaches to demonstrate that the serotonin1A receptor undergoes clathrin-mediated endocytosis upon agonist stimulation and subsequently traffics along the endosomal recycling pathway. In this work, we show that statin-induced chronic cholesterol depletion switches the endocytic pathway of the serotonin1A receptor from clathrin- to caveolin-mediated endocytosis. Interestingly, under these conditions, a significant proportion of endocytosed receptors is rerouted toward lysosomal degradation. To the best of our knowledge, these results constitute one of the first comprehensive reports on the role of membrane cholesterol in GPCR endocytosis and trafficking. These results are significant in our overall understanding of the modulatory effects of membrane lipids on GPCR endocytosis and trafficking and could provide novel insight in developing therapeutic interventions against neuropsychiatric disorders such as depression.

Role of Actin Cytoskeleton in Dynamics and Function of the Serotonin1A Receptor

G protein-coupled receptors (GPCRs) are important membrane proteins in higher eukaryotes that carry out a vast array of cellular signaling and act as major drug targets. The serotonin1A receptor is a prototypical member of the GPCR family and is implicated in neuropsychiatric disorders such as anxiety and depression, besides serving as an important drug target. With an overall goal of exploring the functional consequence of altered receptor dynamics, in this work, we probed the role of the actin cytoskeleton in the dynamics, ligand binding, and signaling of the serotonin1A receptor. We monitored receptor dynamics utilizing single particle tracking, which provides information on relative distribution of receptors in various diffusion modes in addition to diffusion coefficient. We show here that the short-term diffusion coefficient of the receptor increases upon actin destabilization by cytochalasin D. In addition, analysis of individual trajectories shows that there are changes in relative populations of receptors undergoing various types of diffusion upon actin destabilization. The release of dynamic constraint was evident by an increase in the radius of confinement of the receptor upon actin destabilization. The functional implication of such actin destabilization was manifested as an increase in specific agonist binding and downstream signaling, monitored by measuring reduction in cellular cAMP levels. These results bring out the interdependence of GPCR dynamics with cellular signaling.

Mechanisms of signalling and biased agonism in G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are the largest group of cell surface receptors in humans that signal in response to diverse inputs and regulate a plethora of cellular processes. Hence, they constitute one of the primary drug target classes. Progress in our understanding of GPCR dynamics, activation and signalling has opened new possibilities for selective drug development. A key advancement has been provided by the concept of biased agonism, which describes the ability of ligands acting at the same GPCR to elicit distinct cellular signalling profiles by preferentially stabilizing different active conformational states of the receptor. Application of this concept raises the prospect of 'designer' biased agonists as optimized therapeutics with improved efficacy and/or reduced side-effect profiles. However, this application will require a detailed understanding of the spectrum of drug actions and a structural understanding of the drug-receptor interactions that drive distinct pharmacologies. The recent revolution in GPCR structural biology provides unprecedented insights into ligand binding, conformational dynamics and the control of signalling outcomes. These insights, together with new approaches to multi-dimensional analysis of drug action, are allowing refined classification of drugs according to their pharmacodynamic profiles, which can be linked to receptor structure and predictions of preclinical drug efficacy.

PAC1 Receptors: Shapeshifters in Motion.

Shapeshifters, in common mythology, are entities that can undergo multiple physical transformations. As our understanding of G protein-coupled receptors (GPCRs) has accelerated and been refined over the last two decades, we now understand that GPCRs are not static proteins, but rather dynamic structures capable of moving from one posture to the next, and adopting unique functional characteristics at each transition. This model of GPCR dynamics underlies our current understanding of biased agonism-how different ligands to the same receptor can generate different intracellular signals-and constitutive receptor activity, or the level of unbound basal receptor signaling that can be attenuated by inverse agonists. From information derived from related class B receptors, we have recently modeled the structure and molecular dynamics of the full-length pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1)-selective PAC1 receptor (PAC1R, Adcyap1r1). The class B receptors are different from the class A GPCRs in part from the presence of a large extracellular domain (ECD); the transitions of the ECD along with the dynamics of the transmembrane domains (TMD or 7TM) of the PAC1R describes a series of open- and closed-state conformations that appear to identify the mechanisms for receptor activation. The PAC1R shapeshifts also have the ability of delineating the mechanisms and the design of reagents that may direct biased agonism (or antagonism) for potential therapeutics.

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F6.52. Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for drug discovery.