Trace amine-associated receptors (TAARs) are vertebrate olfactory receptors. However, ligand recognition properties of TAARs remain poorly understood, as most are “orphan receptors” without known ...agonists. Here, we identify the first ligands for many rodent TAARs and classify these receptors into two subfamilies based on the phylogeny and binding preference for primary or tertiary amines. Some mouse and rat orthologs have similar response profiles, although independent Taar7 gene expansions led to highly related receptors with altered ligand specificities. Using chimeric TAAR7 receptors, we identified an odor contact site in transmembrane helix III that functions as a selectivity filter. Homology models based on the β2 adrenergic receptor structure indicate spatial proximity of this site to the ligand. Gain-of-function mutations at this site created olfactory receptors with radically altered odor recognition properties. These studies provide new TAAR ligands, valuable tools for studying receptor function, and general insights into the molecular pharmacology of G protein-coupled receptors.
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-HT
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.
G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most ...extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}), a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.
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-HT
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.
Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting; thus GCGR plays an important role in glucose homeostasis. Here we report ...the crystal structure of the seven transmembrane helical domain of human GCGR at 3.4 A resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its native ligand. Beyond the shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors with a large ligand-binding pocket and the first transmembrane helix having a 'stalk' region that extends three alpha- helical turns above the plane of the membrane. The stalk positions the extracellular domain (12kilodaltons) relative to the membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon's amino terminus into the seven transmembrane domain.
Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting, thus GCGR plays an important role in glucose homeostasis. Here we report ...the crystal structure of the seven transmembrane (7TM) helical domain of human GCGR at 3.4 Å resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its natural ligand. Beyond the shared 7TM fold, the GCGR transmembrane domain deviates from class A G protein-coupled receptors with a large ligand binding pocket and the first transmembrane helix having a “stalk” region that extends three alpha-helical turns above the plane of the membrane. The stalk orients the extracellular domain (~12 kDa) relative to the membrane to form the glucagon binding site that captures the peptide and facilitates the insertion of glucagon’s N-terminus into the 7TM domain.
Structural studies of G protein-coupled receptors (GPCRs) are finally providing key mechanistic insights into how these important receptors work. One exciting area of discovery related to drug abuse ...is studies of receptors that recognize and respond to hallucinogenic molecules. The psychedelic drugs lysergic acid diethylamide (LSD) and Salvia (Salvinorin A, SalA) are known to act through G protein-coupled 5-Hydroxytryptamine (5-HT, serotonin)- and opioid receptors, respectively. However, the molecular details of drug action at these receptors remains poorly understood. Structures of all three canonical opioid receptors δ, μ, and κ, reveal key insights into the architecture and ligand binding pocket composition of opioid receptors. These studies also provide a structural basis for opioid selectivity and show why drugs, such as the hallucinogen SalA, are highly specific for their respective target receptor. Water molecules mediating key opioid receptor-ligand interactions further indicate unsatisfied receptor binding and suggest how to develop opioid drugs with better pharmacological profiles and less off-target effects. Pharmacological studies indicate that LSD elicits markedly different signaling at 5-HT receptors compared to the endogenous agonist 5-HT, and acts as a full agonist, β-arrestin biased agonist or even antagonist at different 5-HT receptors. Crystal structures of 5-HT receptors bound to LSD derivatives additionally reveal the binding mode, important receptor-ligand interactions, and the structural basis for the different signaling properties of LSD at different 5-HT receptors. Moreover, serial femtosecond x-ray crystallography (SFX) of the serotonin receptor 5-HT2B bound to an LSD derivative reveals important differences from structural studies on cryo-cooled samples, such as different B-factors and side chain conformations. The high quality room temperature structural data that is devoid of radiation damage likely represents a more accurate receptor conformational ensemble closer to native conditions. Additional structures of the β2-adrenergic receptor (β 2AR) bound to two inverse agonists and an antagonist show how highly specific receptor interactions modulate inhibition of β2AR and probably other GPCRs. Together with docking and mutagenesis studies, the structures presented in this study provide comprehensive structural insights into the mechanism of drug action at GPCRs, which may greatly facilitate the development of safer and more effective therapeutics.
The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a β propeller and an ...α-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC. PUBLICATION ABSTRACT
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