Wang C, Wu H, Katritch V et al.Structure of the human smoothened receptor bound to an antitumour agent. Nature 497:338-343

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Nature (Impact Factor: 41.46). 05/2013; 497(7449). DOI: 10.1038/nature12167
Source: PubMed


The smoothened (SMO) receptor, a key signal transducer in the hedgehog signalling pathway, is responsible for the maintenance of normal embryonic development and is implicated in carcinogenesis. It is classified as a class frizzled (class F) G-protein-coupled receptor (GPCR), although the canonical hedgehog signalling pathway involves the GLI transcription factors and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure of the transmembrane domain of the human SMO receptor bound to the small-molecule antagonist LY2940680 at 2.5 Å resolution. Although the SMO receptor shares the seven-transmembrane helical fold, most of the conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulphide bonds. The ligand binds at the extracellular end of the seven-transmembrane-helix bundle and forms extensive contacts with the loops.

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    • "GPCRs have been solved in different states: inactive (with antagonist bound), fully active (with both agonist and G-protein or surrogate bound), and intermediate active (with agonist but not G-protein or surrogate bound) (see (Venkatakrishnan et al., 2013; Katritch et al., 2013; Granier & Kobilka, 2012; Cooke et al., 2015) for review). With respect to other GPCR classes, the relatively small number of crystallized structures of GPCRs other than class A precludes drawing general structural trends, although some comparative relationships have been established (Hollenstein et al., 2014; Wu et al., 2014; Wang et al., 2013; Cooke et al., 2015). "
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    ABSTRACT: A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, β2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the μ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration.
    Journal of Structural Biology 11/2015; 192(3). DOI:10.1016/j.jsb.2015.10.019 · 3.23 Impact Factor
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    • "Muscarinic (4DAJ (Kruse et al., 2012)), Neurotensin NTSR1 (4GRV (White et al., 2012)), μ receptor (4DKL (Manglik et al., 2012)), δ receptor (4EJ4 (Granier et al., 2012)), κ receptor (4DJH (Wu et al., 2012)), NOP receptor (4EA3 (Thompson et al., 2012)), Protease activated receptor 1 (3VW7 (Zhang et al., 2012)), 5HT 1B (4IAR (Wang et al., 2013a)), 5HT 2B (4IB4 (Wacker et al., 2013)), SMO (4JKV (Wang et al., 2013b)), Glucagon (4L6R (Siu et al., 2013)), CRF1R (4K5Y (Hollenstein et al., 2013)), Chemokine CCR5 (4MBS (Tan et al., 2013)), mGluR1 (4OR2 (Wu et al., 2014)), P2Y12 (4NTJ (Zhang et al., 2014)), mGluR 5 (4OO9 (Dore et al., 2014)), GPR40/FFAR1 (4PHU (Srivastava et al., 2014)), and Orexin OX2R (4S0V (Yin et al., 2014)). "
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    ABSTRACT: Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmacology 05/2015; 763. DOI:10.1016/j.ejphar.2015.05.012 · 2.53 Impact Factor
    • "The main technological advancements that enabled structural studies of GPCRs include (i) development of methods for heterologous expression in insect cells, (ii) discovery of a new method of membrane protein crystallization in lipidic cubic phase (Landau & Rosenbusch, 1996) and subsequent development of tools, instruments, and protocols for automation of crystallization and crystal detection, (iii) development of receptor stabilization technologies, including antibodies toward the intracellular side of GPCRs in Fab form (Rasmussen et al., 2007) and insertion of the T4 phage lysozyme domain (T4L) in the third intracellular (i3) loop (Cherezov et al., 2007; Rosenbaum et al., 2007), and (iv) development of microcrystallography. This toolbox for GPCR crystallization has rapidly expanded with (i) development of an alanine scanning mutagenesis approach enabling receptor stabilization in different functional states Serrano-Vega, Magnani, Shibata, & Tate, 2008; Warne et al., 2008), (ii) identification of other exogenous soluble fusion domains such as the thermostabilized apocytochrome b562RIL (BRIL) (Chun et al., 2012; Liu et al., 2012) and the rubredoxin (Tan et al., 427 The Ion Channel-Coupled Receptor Assay 2013), (iii) introduction of smaller camelid antibodies called nanobodies (Rasmussen, DeVree, et al., 2011a), and (iv) insertion of soluble domains in the N-terminus (Fenalti et al., 2014; Rasmussen, Choi, et al., 2011; Siu et al., 2013; Wang et al., 2013; Wu et al., 2014) and in the i2 loop (Hollenstein et al., 2013). Alone or in combination, these methods led to the structural determination of 25 other GPCRs from four different classes over the past 7 years. "
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    ABSTRACT: Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"uncoupled" receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L). © 2015 Elsevier Inc. All rights reserved.
    Membrane Proteins—Production and Functional Characterization, 03/2015: chapter 20: pages 425-54;
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