G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the ...transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.
Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We ...previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.
The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this ...benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.
We investigate the X-ray and UV emission detected by RHESSI and TRACE in the context of a solar flare on the 16th November 2002 with the goal of better understanding the evolution of the flare. We ...analysed the characteristics of the X-ray emission in the 12–25 and 25–50 keV energy range while we looked at the UV emission at 1600 Å . The flare appears to have two distinct phases of emission separated by a 25-s time delay, with the first phase being energetically more important. We found good temporal and spatial agreement between the 25–50 keV X-rays and the most intense areas of the 1600 Å UV emission. We also observed an extended 100-arcsec < 25 keV source that appears coronal in nature and connects two separated UV ribbons later in the flare. Using the observational properties in X-ray and UV wavelengths, we propose two explanations for the flare evolution in relation to the spine/fan magnetic field topology and the accelerated electrons. We find that a combination of quasi separatrix layer reconnection and null-point reconnection is required to account for the observed properties of the X-ray and UV emission.
Coronal mass ejections (CMEs) are one of the primary manifestations of solar activity and can drive severe space weather effects. Therefore, it is vital to work towards being able to predict their ...occurrence. However, many aspects of CME formation and eruption remain unclear, including whether magnetic flux ropes are present before the onset of eruption and the key mechanisms that cause CMEs to occur. In this work, the pre-eruptive coronal configuration of an active region that produced an interplanetary CME with a clear magnetic flux rope structure at 1 AU is studied. A forward-S sigmoid appears in extreme-ultraviolet (EUV) data two hours before the onset of the eruption (SOL2012-06-14), which is interpreted as a signature of a right-handed flux rope that formed prior to the eruption. Flare ribbons and EUV dimmings are used to infer the locations of the flux rope footpoints. These locations, together with observations of the global magnetic flux distribution, indicate that an interaction between newly emerged magnetic flux and pre-existing sunspot field in the days prior to the eruption may have enabled the coronal flux rope to form via tether-cutting-like reconnection. Composition analysis suggests that the flux rope had a coronal plasma composition, supporting our interpretation that the flux rope formed via magnetic reconnection in the corona. Once formed, the flux rope remained stable for two hours before erupting as a CME.
The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently
. Of these, MRGPRX2 and MRGPRX4 are key physiological and ...pathological mediators of itch and related mast cell-mediated hypersensitivity reactions
. MRGPRX2 couples to both G
and G
in mast cells
. Here we describe agonist-stabilized structures of MRGPRX2 coupled to G
and G
in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G
. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using ...the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq-coupled M3-DREADD within the same neuronal population facilitated the sequential and bidirectional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators.
•Structure-guided approach for κ-opioid receptor (KOR)-DREADD (KORD) design•KORD is selectively activated by salvinorin B, and not by endogenous opioids•KORD robustly silenced multiple neuronal subtypes•Inhibitory KORD combined with excitatory hM3Dq for multiplexed behavioral control
The κ-opioid receptor (KOR) was used as a template to generate a novel inhibitory DREADD (KORD), which is activated by salvinorin B and insensitive to endogenous opioid peptides. Sequential activation of the inhibitory KOR-DREADD and an excitatory M3-DREADD facilitated the bidirectional, multiplexed modulation of behavior.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the ...regulation of blood pressure and core body temperature, oncogenesis, and immune function. Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness by activating two high-affinity G-proteincoupled receptors, type 1A (MT1) and type 1B (MT2). Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids, and is one of the most popular supplements in the United States. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine. The structure of MT2 is described in an accompanying paper. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
The human MT1 and MT2 melatonin receptors are G-proteincoupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns. Drug development efforts have targeted both receptors for ...the treatment of insomnia, circadian rhythm and mood disorders, and cancer, and MT2 has also been implicated in type 2 diabetes. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in 3Hmelatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.
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