Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Ligand and G-protein selectivity in the κ-opioid receptor.

The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Molecular basis for selective activation of DREADD-based chemogenetics.

Designer receptors exclusively activated by designer drugs (DREADDs) represent a powerful chemogenetic technology for the remote control of neuronal activity and cellular signalling1-4. The muscarinic receptor-based DREADDs are the most widely used chemogenetic tools in neuroscience research. The Gq-coupled DREADD (hM3Dq) is used to enhance neuronal activity, whereas the Gi/o-coupled DREADD (hM4Di) is utilized to inhibit neuronal activity5. Here we report four DREADD-related cryogenic electron microscopy high-resolution structures: a hM3Dq-miniGq complex and a hM4Di-miniGo complex bound to deschloroclozapine; a hM3Dq-miniGq complex bound to clozapine-N-oxide; and a hM3R-miniGq complex bound to iperoxo. Complemented with mutagenesis, functional and computational simulation data, our structures reveal key details of the recognition of DREADD chemogenetic actuators and the molecular basis for activation. These findings should accelerate the structure-guided discovery of next-generation chemogenetic tools.

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 recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq 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 Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

Ligand recognition and allosteric modulation of the human MRGPRX1 receptor.

The human MAS-related G protein-coupled receptor X1 (MRGPRX1) is preferentially expressed in the small-diameter primary sensory neurons and involved in the mediation of nociception and pruritus. Central activation of MRGPRX1 by the endogenous opioid peptide fragment BAM8-22 and its positive allosteric modulator ML382 has been shown to effectively inhibit persistent pain, making MRGPRX1 a promising target for non-opioid pain treatment. However, the activation mechanism of MRGPRX1 is still largely unknown. Here we report three high-resolution cryogenic electron microscopy structures of MRGPRX1-Gαq in complex with BAM8-22 alone, with BAM8-22 and ML382 simultaneously as well as with a synthetic agonist compound-16. These structures reveal the agonist binding mode for MRGPRX1 and illuminate the structural requirements for positive allosteric modulation. Collectively, our findings provide a molecular understanding of the activation and allosteric modulation of the MRGPRX1 receptor, which could facilitate the structure-based design of non-opioid pain-relieving drugs.

Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.

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) Gαßγ 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 Gαßγ 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.

Molecular glues as potential GPCR therapeutics.

"Molecular Glues" are defined as small molecules that can either be endogenous or synthetic which promote interactions between proteins at their interface. Allosteric modulators, specifically GPCR allosteric modulators, can promote both the association and the dissociation of a given receptor's transducer but accomplishes this "at a distance" from the interface. However, recent structures of GPCR G protein complexes in the presence of allosteric modulators indicate that some GPCR allosteric modulators can act as "molecular glues" interacting with both the receptor and the transducer at the interface biasing transducer signaling in both a positive and negative manner depending on the transducer. Given these phenomena we discuss the implications for this class of allosteric modulators to be used as molecular tools and for future drug development.

CryoEM structures of adhesion in GPCR CD97: Filling in some of the gaps.

CD97 (ADGRE5) is an adhesion GPCR (aGPCR) that plays crucial roles in the immune system and cancer. In this issue of Cell Chemical Biology, Wang et al.1 present the cryoEM structures of CD97 in complex with G13, Gq, and Gs G protein subtypes, revealing in-depth insight into aGPCR activation and G protein selectivity.

Structural genomics of the human dopamine receptor system.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

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.

Inactive and active state structures template selective tools for the human 5-HT5A receptor.

Serotonin receptors are important targets for established therapeutics and drug development as they are expressed throughout the human body and play key roles in cell signaling. There are 12 serotonergic G protein-coupled receptor members encoded in the human genome, of which the 5-hydroxytryptamine (5-HT)5A receptor (5-HT5AR) is the least understood and lacks selective tool compounds. Here, we report four high-resolution (2.73-2.80 Å) structures of human 5-HT5ARs, including an inactive state structure bound to an antagonist AS2674723 by crystallization and active state structures bound to a partial agonist lisuride and two full agonists, 5-carboxamidotryptamine (5-CT) and methylergometrine, by cryo-EM. Leveraging the new structures, we developed a highly selective and potent antagonist for 5-HT5AR. Collectively, these findings both enhance our understanding of this enigmatic receptor and provide a roadmap for structure-based drug discovery for 5-HT5AR.

Signaling snapshots of a serotonin receptor activated by the prototypical psychedelic LSD.

Serotonin (5-hydroxytryptamine [5-HT]) 5-HT2-family receptors represent essential targets for lysergic acid diethylamide (LSD) and all other psychedelic drugs. Although the primary psychedelic drug effects are mediated by the 5-HT2A serotonin receptor (HTR2A), the 5-HT2B serotonin receptor (HTR2B) has been used as a model receptor to study the activation mechanisms of psychedelic drugs due to its high expression and similarity to HTR2A. In this study, we determined the cryo-EM structures of LSD-bound HTR2B in the transducer-free, Gq-protein-coupled, and ß-arrestin-1-coupled states. These structures provide distinct signaling snapshots of LSD's action, ranging from the transducer-free, partially active state to the transducer-coupled, fully active states. Insights from this study will both provide comprehensive molecular insights into the signaling mechanisms of the prototypical psychedelic LSD and accelerate the discovery of novel psychedelic drugs.

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.

Structure and dynamics of a constitutively active neurotensin receptor.

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae.

Type II secretion systems consist of an assembly of 12-15 Gsp proteins responsible for transporting a variety of virulence factors across the outer membrane in several pathogenic bacteria. In Vibrio cholerae, the major virulence factor cholera toxin is secreted by the Eps Type II secretion apparatus consisting of 14 Eps proteins. One of these, EpsE, is a cytoplasmic putative NTPase essential for the functioning of the Eps system and member of the GspE subfamily of Type II secretion ATPases. The crystal structure of a truncated form of EpsE in nucleotide-liganded and unliganded state has been determined, and reveals a two-domain architecture with the four characteristic sequence "boxes" of the GspE subfamily clustering around the nucleotide-binding site of the C-domain. This domain contains two C-terminal subdomains not reported before in this superfamily of NTPases. One of these subdomains contains a four-cysteine motif that appears to be involved in metal binding as revealed by anomalous difference density. The EpsE subunits form a right-handed helical arrangement in the crystal with extensive and conserved contacts between the C and N domains of neighboring subunits. Combining the most conserved interface with the quaternary structure of the C domain in a distant homolog, a hexameric model for EpsE is proposed which may reflect the assembly of this critical protein in the Type II secretion system. The nucleotide ligand contacts both domains in this model. The N2-domain-containing surface of the hexamer appears to be highly conserved in the GspE family and most likely faces the inner membrane interacting with other members of the Eps system.