Vsevolod Katritch

Structure of the human glucagon class B G-protein-coupled receptor

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 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 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 (∼12 kilodaltons) 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.

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.

Regulating Migration The migration of cells around the body is an important factor in cancer development and the establishment of infection. Movement is induced by small proteins called chemokines, and so for a specific function, migration is controlled by a relevant chemokine binding to its respective receptor. This family of receptors is known as guanine (G) protein–coupled receptors, which span cell membranes to mediate between external signals from chemokines and internal mechanisms. The chemokine receptor CXCR4 is implicated in many types of cancer and in infection, and Wu et al. (p. 1066, published online 7 October; see the Report by Chien et al.) report on a series of crystal structures obtained for CXCR4 bound to small molecules. In every case, the same homodimer structure was observed, suggesting that the interface is functionally relevant. These structures offer insights into the interactions between CXCR4 and its natural chemokine, as well as with the virus HIV-1. Five crystal structures provide insight into chemokine and HIV-1 recognition. Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein–coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein–coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.

Structure of the human dopamine d3 receptor in complex with a d2/d3 selective antagonist.

Tweaking Dopamine Reception Dopamine modulates many cognitive and emotional functions of the human brain by activating G protein–coupled receptors. Antipsychotic drugs that block two of the receptor subtypes are used to treat schizophrenia but have multiple side effects. Chien et al. (p. 1091; see the Research Article by Wu et al.) resolved the crystal structure of one receptor in complex with a small-molecule inhibitor at 3.15 angstrom resolution. Homology modeling with other receptor subtypes might be a promising route to reveal potential structural differences that can be exploited in the design of selective therapeutic inhibitors having fewer side effects. Discovery of a binding site in the extracellular domain of a dopamine receptor offers hope for more selective therapeutics. Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein–coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

Structure of an Agonist-Bound Human A2A Adenosine Receptor

Changes associated with conformationally selective agonist binding shed light on G protein–coupled receptor activation. Activation of G protein–coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A2A adenosine receptor (A2AAR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A2AAR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A2AAR and its ligand. The results define the molecule UK-432097 as a “conformationally selective agonist” capable of receptor stabilization in a specific active-state configuration.

Structure of the human κ-opioid receptor in complex with JDTic

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.

Structure-Function of the G Protein–Coupled Receptor Superfamily

During the past few years, crystallography of G protein-coupled receptors (GPCRs) has experienced exponential growth, resulting in the determination of the structures of 16 distinct receptors-9 of them in 2012 alone. Including closely related subtype homology models, this coverage amounts to approximately 12% of the human GPCR superfamily. The adrenergic, rhodopsin, and adenosine receptor systems are also described by agonist-bound active-state structures, including a structure of the receptor-G protein complex for the β(2)-adrenergic receptor. Biochemical and biophysical techniques, such as nuclear magnetic resonance and hydrogen-deuterium exchange coupled with mass spectrometry, are providing complementary insights into ligand-dependent dynamic equilibrium between different functional states. Additional details revealed by high-resolution structures illustrate the receptors as allosteric machines that are controlled not only by ligands but also by ions, lipids, cholesterol, and water. This wealth of data is helping redefine our knowledge of how GPCRs recognize such a diverse array of ligands and how they transmit signals 30 angstroms across the cell membrane; it also is shedding light on a structural basis of GPCR allosteric modulation and biased signaling.

Structure of the human histamine H1 receptor complex with doxepin.

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H1 receptor (H1R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H1R complex with doxepin, a first-generation H1R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 4286.48, a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H1R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 1915.39 and/or Lys 179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H1R antagonist specificity against H1R.

Structural basis for allosteric regulation of GPCRs by sodium ions.

GPCR Close-Up Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232) used a protein-engineering strategy to produce a stabilized version of the human A2Aadenosine receptor (A2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na+ on ligand binding and how cholesterol may contribute to GPCR stabilization. A protein-engineering strategy yields a closer look at the receptor-bound water, sodium, and lipid molecules. Pharmacological responses of G protein–coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A2A adenosine receptor by replacing its third intracellular loop with apocytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp2.50. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

Biased signaling pathways in β2-adrenergic receptor characterized by 19f-nmr

Choosing a Path The β2-adrenergic receptor (β2AR) is a G protein–coupled receptor that recognizes diverse ligands to trigger signaling in the cell. Besides binding G proteins, activated β2AR can be phosphorylated and bind arrestin, which redirects signaling to other pathways. Some β2AR ligands are “biased” in that they differentially activate G protein or arrestin signaling. Liu et al. (p. 1106, published online 19 January; see the Perspective by Sprang and Chief Elk) used 19F-NMR spectroscopy to examine conformational changes associated with a range of ligands and discovered that biased ligands caused differential shifts in equilibrium between two conformational states—the G protein binding state and the arrestin binding state—and thus provide a basis for rational design of pharmacological ligands. Selective effects of different ligands provide insights into the structural plasticity of receptor signaling. Extracellular ligand binding to G protein–coupled receptors (GPCRs) modulates G protein and β-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific 19F-NMR (fluorine-19 nuclear magnetic resonance) labels in the β2-adrenergic receptor (β2AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium toward the G protein–specific active state of helix VI. In contrast, β-arrestin–biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of β2AR in partial and biased agonist signaling.clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): May 17, 2012 www.sciencemag.org (this information is current as of The following resources related to this article are available online at

Structural features for functional selectivity at serotonin receptors.

Dissecting Serotonin Receptors Serotonin receptors are the targets for many widely used drugs prescribed to treat ailments from depression to obesity and migraine headaches (see the Perspective by Palczewski and Kiser). C. Wang et al. (p. 610, published online 21 March) and Wacker et al. (p. 615, published online 21 March) describe crystal structures of two members of the serotonin family of receptors bound to antimigraine medications or to a precursor of the hallucinogenic drug LSD. Subtle differences in the way particular ligands bind to the receptors cause substantial differences in the signals generated by the receptor and the consequent biological responses. The structures reveal how the same ligand can activate one or both of the two main serotonin receptor signaling mechanisms, depending on which particular receptor it binds. Structures of serotonin receptor family members in complex with the fungal alkaloid ergot offer clues for drug designers. [Also see Perspective by Palczewski and Kiser] Drugs active at G protein–coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-HT2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT2B receptor bound to ERG and compared it with the 5-HT1B/ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities.