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"Reid, H."
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TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome
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.
Development of BRET sensors for nearly all major G proteins show that GPCR–G-protein coupling ranges from promiscuous to extremely specific, Switch III is a novel site for G-protein engineering, and optimal donor–acceptor positioning is non-obvious.
Journal Article
Nanobody-enabled monitoring of kappa opioid receptor states
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.
Recent studies revealed that G protein-coupled receptors rapidly interconvert between multiple states. Here, authors use the kappa opioid receptor (KOR) and show how two state-dependent nanobodies provide real-time reporting of ligand stabilized states with KOR and other GPCRs.
Journal Article
Molecular mechanism of biased signaling at the kappa opioid receptor
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.
Biased signaling in κ-opiod receptors (KOR) offer an attractive strategy for pain management. Here the authors identify determinants of KOR signaling bias using structural methods in combination with molecular dynamics simulations.
Journal Article
Structure, function and pharmacology of human itch GPCRs
The MRGPRX family of receptors (MRGPRX1–4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently
1
. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions
2
–
5
. MRGPRX2 couples to both G
i
and G
q
in mast cells
6
. Here we describe agonist-stabilized structures of MRGPRX2 coupled to G
i1
and G
q
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
q
. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
Structural studies of the itch receptors MRGPRX2 and MRGPRX4 in complex with endogenous and synthetic ligands provide a basis for the development of therapeutic compounds for pain, itch and mast cell-mediated hypersensitivity.
Journal Article
On-Disc Observations of Flux Rope Formation Prior to Its Eruption
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.
Journal Article
Structural basis of ligand recognition at the human MT1 melatonin receptor
Melatonin (
N
-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms
1
by synchronization to environmental cues and is involved in diverse physiological processes
2
such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function
3
. Melatonin is formed in the pineal gland in a light-regulated manner
4
by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness
5
by activating two high-affinity G-protein-coupled receptors, type 1A (MT
1
) and type 1B (MT
2
)
3
,
6
. 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
7
. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids
8
,
9
, and is one of the most popular supplements in the United States
10
. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT
1
in complex with four agonists: the insomnia drug ramelteon
11
, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine
12
,
13
. The structure of MT
2
is described in an accompanying paper
14
. Although the MT
1
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 MT
1
, 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 MT
1
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 MT
1
melatonin receptor differs markedly from 5-HT receptors and shows atypical ligand entry; its structure with various ligands sheds light on receptor specificity.
Journal Article
Structural determinants of 5-HT2B receptor activation and biased agonism
Serotonin (5-hydroxytryptamine; 5-HT) receptors modulate a variety of physiological processes ranging from perception, cognition and emotion to vascular and smooth muscle contraction, platelet aggregation, gastrointestinal function and reproduction. Drugs that interact with 5-HT receptors effectively treat diseases as diverse as migraine headaches, depression and obesity. Here we present four structures of a prototypical serotonin receptor—the human 5-HT2B receptor—in complex with chemically and pharmacologically diverse drugs, including methysergide, methylergonovine, lisuride and LY266097. A detailed analysis of these structures complemented by comprehensive interrogation of signaling illuminated key structural determinants essential for activation. Additional structure-guided mutagenesis experiments revealed binding pocket residues that were essential for agonist-mediated biased signaling and β-arrestin2 translocation. Given the importance of 5-HT receptors for a large number of therapeutic indications, insights derived from these studies should accelerate the design of safer and more effective medications.
Journal Article
Bidirectional phonon emission in two-dimensional heterostructures triggered by ultrafast charge transfer
Photoinduced charge transfer in van der Waals heterostructures occurs on the 100 fs timescale despite weak interlayer coupling and momentum mismatch. However, little is understood about the microscopic mechanism behind this ultrafast process and the role of the lattice in mediating it. Here, using femtosecond electron diffraction, we directly visualize lattice dynamics in photoexcited heterostructures of WSe
2
/WS
2
monolayers. Following the selective excitation of WSe
2
, we measure the concurrent heating of both WSe
2
and WS
2
on a picosecond timescale—an observation that is not explained by phonon transport across the interface. Using first-principles calculations, we identify a fast channel involving an electronic state hybridized across the heterostructure, enabling phonon-assisted interlayer transfer of photoexcited electrons. Phonons are emitted in both layers on the femtosecond timescale via this channel, consistent with the simultaneous lattice heating observed experimentally. Taken together, our work indicates strong electron–phonon coupling via layer-hybridized electronic states—a novel route to control energy transport across atomic junctions.
Femtosecond electron diffraction and ab initio theory unravel ultrafast lattice dynamics in photoexcited two-dimensional heterostructures during charge transfer.
Journal Article