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  • 1
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    Structural flexibility of the G{alpha}s {alpha}-helical domain in the {beta}2-adrenoceptor Gs complex [Pharmacology] (2011)
    National Academy of Sciences
    Details
    Publication Date: 2011-09-21
    Description: The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β2-adrenoceptor (β2AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 2
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    Two-metal-Ion catalysis in adenylyl cyclase (1999)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 1999-07-31
    Description: Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tesmer, J J -- Sunahara, R K -- Johnson, R A -- Gosselin, G -- Gilman, A G -- Sprang, S R -- DK38828/DK/NIDDK NIH HHS/ -- DK46371/DK/NIDDK NIH HHS/ -- GM34497/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1999 Jul 30;285(5428):756-60.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute, Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9050, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/10427002" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphate/metabolism ; Adenylyl Cyclase Inhibitors ; Adenylyl Cyclases/chemistry/genetics/*metabolism ; Animals ; Aspartic Acid/metabolism ; Binding Sites ; Catalysis ; Crystallography, X-Ray ; Deoxyadenine Nucleotides/metabolism/pharmacology ; Dideoxynucleotides ; Dimerization ; Enzyme Inhibitors/metabolism ; Hydrogen Bonding ; Ligands ; Magnesium/*metabolism ; Manganese/*metabolism ; Models, Molecular ; Mutation ; Protein Conformation ; Protein Folding ; Rats ; Thionucleotides/metabolism/pharmacology ; Zinc/*metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 3
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    Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS (1998)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 1998-01-07
    Description: The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein alpha subunit (Gsalpha) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by Gsalpha.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Tesmer, J J -- Sunahara, R K -- Gilman, A G -- Sprang, S R -- DK38828/DK/NIDDK NIH HHS/ -- DK46371/DK/NIDDK NIH HHS/ -- GM34497/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1997 Dec 12;278(5345):1907-16.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75235-9050, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/9417641" target="_blank"〉PubMed〈/a〉
    Keywords: Adenosine Triphosphate/metabolism ; Adenylyl Cyclase Inhibitors ; Adenylyl Cyclases/*chemistry/metabolism ; Amino Acid Sequence ; Binding Sites ; Catalysis ; Colforsin/metabolism ; Crystallization ; Crystallography, X-Ray ; Dimerization ; Enzyme Activation ; GTP-Binding Protein alpha Subunits, Gs/*chemistry/metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/*chemistry/metabolism ; Ligands ; Models, Molecular ; Molecular Sequence Data ; Mutation ; Phosphorylation ; Protein Conformation ; Protein Structure, Secondary ; Protein Structure, Tertiary
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 4
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    Crystal structure of the adenylyl cyclase activator Gsalpha (1998)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 1998-01-07
    Description: The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sunahara, R K -- Tesmer, J J -- Gilman, A G -- Sprang, S R -- DK46371/DK/NIDDK NIH HHS/ -- GM34497/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1997 Dec 12;278(5345):1943-7.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/9395396" target="_blank"〉PubMed〈/a〉
    Keywords: Adenylyl Cyclases/chemistry/*metabolism ; Amino Acid Sequence ; Binding Sites ; Conserved Sequence ; Crystallization ; Crystallography, X-Ray ; Dimerization ; Enzyme Activation ; GTP Phosphohydrolases/metabolism ; GTP-Binding Protein alpha Subunits, Gi-Go/chemistry/metabolism ; GTP-Binding Protein alpha Subunits, Gs/*chemistry/metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/*chemistry/metabolism ; Guanosine Triphosphate/metabolism ; Hydrolysis ; Magnesium/metabolism ; Models, Molecular ; Molecular Sequence Data ; *Protein Conformation ; Protein Structure, Secondary ; Signal Transduction
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 5
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    Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor (2011)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2011-01-14
    Description: G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human beta(2) adrenergic receptor (beta(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive beta(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 A outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058308/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058308/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Rasmussen, Soren G F -- Choi, Hee-Jung -- Fung, Juan Jose -- Pardon, Els -- Casarosa, Paola -- Chae, Pil Seok -- Devree, Brian T -- Rosenbaum, Daniel M -- Thian, Foon Sun -- Kobilka, Tong Sun -- Schnapp, Andreas -- Konetzki, Ingo -- Sunahara, Roger K -- Gellman, Samuel H -- Pautsch, Alexander -- Steyaert, Jan -- Weis, William I -- Kobilka, Brian K -- GM083118/GM/NIGMS NIH HHS/ -- GM56169/GM/NIGMS NIH HHS/ -- NS028471/NS/NINDS NIH HHS/ -- P01 GM75913/GM/NIGMS NIH HHS/ -- P60DK-20572/DK/NIDDK NIH HHS/ -- R01 GM068603/GM/NIGMS NIH HHS/ -- R01 GM083118/GM/NIGMS NIH HHS/ -- R01 GM083118-04/GM/NIGMS NIH HHS/ -- R37 NS028471/NS/NINDS NIH HHS/ -- R37 NS028471-21/NS/NINDS NIH HHS/ -- England -- Nature. 2011 Jan 13;469(7329):175-80. doi: 10.1038/nature09648.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21228869" target="_blank"〉PubMed〈/a〉
    Keywords: Adrenergic beta-2 Receptor ; Agonists/*chemistry/immunology/metabolism/*pharmacology ; Animals ; Binding Sites ; Camelids, New World ; Crystallography, X-Ray ; Drug Inverse Agonism ; Humans ; Immunoglobulin Fragments/*chemistry/*immunology/metabolism/pharmacology ; Ligands ; Models, Molecular ; Movement/drug effects ; Nanostructures/*chemistry ; Opsins/agonists/chemistry/metabolism ; Propanolamines/chemistry/metabolism/pharmacology ; Protein Conformation/drug effects ; Protein Stability/drug effects ; Receptors, Adrenergic, beta-2/*chemistry/*metabolism ; Viral Proteins/chemistry/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 6
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    Conformational changes in the G protein Gs induced by the beta2 adrenergic receptor (2011)
    Nature Publishing Group (NPG)
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    Publication Date: 2011-10-01
    Description: G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein alpha-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human beta(2) adrenergic receptor (beta(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the beta(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the beta(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the alpha-subunit of Gs and consequently alters the 'P-loop' that binds the beta-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and beta-phosphate coordination are key determinants of GDP (and GTP) binding affinity.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448949/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448949/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Chung, Ka Young -- Rasmussen, Soren G F -- Liu, Tong -- Li, Sheng -- DeVree, Brian T -- Chae, Pil Seok -- Calinski, Diane -- Kobilka, Brian K -- Woods, Virgil L Jr -- Sunahara, Roger K -- AI076961/AI/NIAID NIH HHS/ -- AI081982/AI/NIAID NIH HHS/ -- AI2008031/AI/NIAID NIH HHS/ -- CA118595/CA/NCI NIH HHS/ -- GM008270/GM/NIGMS NIH HHS/ -- GM066170/GM/NIGMS NIH HHS/ -- GM068603/GM/NIGMS NIH HHS/ -- GM083118/GM/NIGMS NIH HHS/ -- GM093325/GM/NIGMS NIH HHS/ -- GM20501/GM/NIGMS NIH HHS/ -- HL071078/HL/NHLBI NIH HHS/ -- NS28471/NS/NINDS NIH HHS/ -- P60DK-20572/DK/NIDDK NIH HHS/ -- R01 GM020501/GM/NIGMS NIH HHS/ -- R01 GM068603/GM/NIGMS NIH HHS/ -- R01 GM068603-03/GM/NIGMS NIH HHS/ -- R01 GM068603-04/GM/NIGMS NIH HHS/ -- R01 GM068603-05/GM/NIGMS NIH HHS/ -- R01 GM083118/GM/NIGMS NIH HHS/ -- R01 GM083118-02/GM/NIGMS NIH HHS/ -- R01 GM083118-03/GM/NIGMS NIH HHS/ -- R01 GM083118-04/GM/NIGMS NIH HHS/ -- RR029388/RR/NCRR NIH HHS/ -- England -- Nature. 2011 Sep 28;477(7366):611-5. doi: 10.1038/nature10488.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21956331" target="_blank"〉PubMed〈/a〉
    Keywords: Adrenergic beta-2 Receptor Agonists/chemistry/metabolism ; Animals ; Biocatalysis ; Catalytic Domain ; Cattle ; Crystallography, X-Ray ; Deuterium Exchange Measurement ; GTP-Binding Protein alpha Subunits, Gs/*chemistry/*metabolism/ultrastructure ; Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/metabolism ; Humans ; Models, Molecular ; Protein Binding ; Protein Conformation ; Receptors, Adrenergic, beta-2/*chemistry/*metabolism/ultrastructure
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 7
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    Crystal structure of the beta2 adrenergic receptor-Gs protein complex (2011)
    Nature Publishing Group (NPG)
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    Publication Date: 2011-07-21
    Description: G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The beta(2) adrenergic receptor (beta(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric beta(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the beta(2)AR and Gs involve the amino- and carboxy-terminal alpha-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the beta(2)AR include a 14 A outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an alpha-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the alpha-helical domain of Galphas relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184188/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184188/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Rasmussen, Soren G F -- DeVree, Brian T -- Zou, Yaozhong -- Kruse, Andrew C -- Chung, Ka Young -- Kobilka, Tong Sun -- Thian, Foon Sun -- Chae, Pil Seok -- Pardon, Els -- Calinski, Diane -- Mathiesen, Jesper M -- Shah, Syed T A -- Lyons, Joseph A -- Caffrey, Martin -- Gellman, Samuel H -- Steyaert, Jan -- Skiniotis, Georgios -- Weis, William I -- Sunahara, Roger K -- Kobilka, Brian K -- GM083118/GM/NIGMS NIH HHS/ -- GM56169/GM/NIGMS NIH HHS/ -- GM75915/GM/NIGMS NIH HHS/ -- NS028471/NS/NINDS NIH HHS/ -- P01 GM75913/GM/NIGMS NIH HHS/ -- P50GM073210/GM/NIGMS NIH HHS/ -- P60DK-20572/DK/NIDDK NIH HHS/ -- R01 GM068603/GM/NIGMS NIH HHS/ -- R01 GM068603-01/GM/NIGMS NIH HHS/ -- R01 GM068603-02/GM/NIGMS NIH HHS/ -- R01 GM068603-03/GM/NIGMS NIH HHS/ -- R01 GM068603-04/GM/NIGMS NIH HHS/ -- R01 GM068603-05/GM/NIGMS NIH HHS/ -- T32-GM008270/GM/NIGMS NIH HHS/ -- U54 GM094599/GM/NIGMS NIH HHS/ -- U54GM094599/GM/NIGMS NIH HHS/ -- England -- Nature. 2011 Jul 19;477(7366):549-55. doi: 10.1038/nature10361.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21772288" target="_blank"〉PubMed〈/a〉
    Keywords: Adrenergic beta-2 Receptor Agonists/chemistry/metabolism ; Animals ; Catalytic Domain ; Cattle ; Crystallization ; Crystallography, X-Ray ; Enzyme Activation ; GTP-Binding Protein alpha Subunits, Gs/*chemistry/*metabolism ; Models, Molecular ; Multiprotein Complexes/chemistry/metabolism ; Protein Binding ; Rats ; Receptors, Adrenergic, beta-2/*chemistry/*metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 8
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    Structure and function of an irreversible agonist-beta(2) adrenoceptor complex (2011)
    Nature Publishing Group (NPG)
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    Publication Date: 2011-01-14
    Description: G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human beta(2) adrenergic receptor (beta(2)AR) as a guide, we designed a beta(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent beta(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound beta(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 A resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 mus) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074335/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074335/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Rosenbaum, Daniel M -- Zhang, Cheng -- Lyons, Joseph A -- Holl, Ralph -- Aragao, David -- Arlow, Daniel H -- Rasmussen, Soren G F -- Choi, Hee-Jung -- Devree, Brian T -- Sunahara, Roger K -- Chae, Pil Seok -- Gellman, Samuel H -- Dror, Ron O -- Shaw, David E -- Weis, William I -- Caffrey, Martin -- Gmeiner, Peter -- Kobilka, Brian K -- 50GM073210/GM/NIGMS NIH HHS/ -- GM56169/GM/NIGMS NIH HHS/ -- GM75915/GM/NIGMS NIH HHS/ -- M083118/PHS HHS/ -- NS028471/NS/NINDS NIH HHS/ -- P01 GM75913/GM/NIGMS NIH HHS/ -- P60DK-20572/DK/NIDDK NIH HHS/ -- R01 GM068603/GM/NIGMS NIH HHS/ -- R37 NS028471/NS/NINDS NIH HHS/ -- R37 NS028471-20/NS/NINDS NIH HHS/ -- England -- Nature. 2011 Jan 13;469(7329):236-40. doi: 10.1038/nature09665.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21228876" target="_blank"〉PubMed〈/a〉
    Keywords: Adrenergic beta-2 Receptor Agonists/*chemistry/*metabolism ; Crystallization ; Crystallography, X-Ray ; Disulfides/chemistry/metabolism ; Drug Inverse Agonism ; Heterotrimeric GTP-Binding Proteins/metabolism ; Humans ; Lipid Bilayers/chemistry/metabolism ; Models, Molecular ; Molecular Dynamics Simulation ; Procaterol/chemistry/metabolism ; Propanolamines/chemistry/metabolism ; Protein Conformation ; Receptors, Adrenergic, beta-2/*chemistry/*metabolism ; Recombinant Fusion Proteins/chemistry/metabolism ; Viral Proteins/chemistry/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 9
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    Crystal structure of the micro-opioid receptor bound to a morphinan antagonist (2012)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2012-03-23
    Description: Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled micro-opioid receptor (micro-OR) in the central nervous system. Here we describe the 2.8 A crystal structure of the mouse micro-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the micro-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3523197/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3523197/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Manglik, Aashish -- Kruse, Andrew C -- Kobilka, Tong Sun -- Thian, Foon Sun -- Mathiesen, Jesper M -- Sunahara, Roger K -- Pardo, Leonardo -- Weis, William I -- Kobilka, Brian K -- Granier, Sebastien -- DA031418/DA/NIDA NIH HHS/ -- NS028471/NS/NINDS NIH HHS/ -- R01 GM083118/GM/NIGMS NIH HHS/ -- R01 NS028471/NS/NINDS NIH HHS/ -- R21 DA031418/DA/NIDA NIH HHS/ -- England -- Nature. 2012 Mar 21;485(7398):321-6. doi: 10.1038/nature10954.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22437502" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Binding Sites ; Crystallography, X-Ray ; Ligands ; Mice ; Models, Molecular ; Morphinans/*chemistry/metabolism/pharmacology ; Protein Conformation ; Protein Multimerization ; Receptors, Opioid, mu/*antagonists & inhibitors/*chemistry/metabolism ; Solvents/chemistry
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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    Conformational biosensors reveal GPCR signalling from endosomes (2013)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2013-03-22
    Description: A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the beta2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835555/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835555/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Irannejad, Roshanak -- Tomshine, Jin C -- Tomshine, Jon R -- Chevalier, Michael -- Mahoney, Jacob P -- Steyaert, Jan -- Rasmussen, Soren G F -- Sunahara, Roger K -- El-Samad, Hana -- Huang, Bo -- von Zastrow, Mark -- DA010711/DA/NIDA NIH HHS/ -- DA012864/DA/NIDA NIH HHS/ -- F32 DA029993/DA/NIDA NIH HHS/ -- GM083118/GM/NIGMS NIH HHS/ -- P01 DA010154/DA/NIDA NIH HHS/ -- P01 NS053709/NS/NINDS NIH HHS/ -- R01 DA012864/DA/NIDA NIH HHS/ -- R01 GM068603/GM/NIGMS NIH HHS/ -- R01 GM083118/GM/NIGMS NIH HHS/ -- R29 DA010711/DA/NIDA NIH HHS/ -- R37 DA010711/DA/NIDA NIH HHS/ -- T32 GM007767/GM/NIGMS NIH HHS/ -- England -- Nature. 2013 Mar 28;495(7442):534-8. doi: 10.1038/nature12000. Epub 2013 Mar 20.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Psychiatry, University of California, San Francisco, California 94158, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23515162" target="_blank"〉PubMed〈/a〉
    Keywords: Adrenergic beta-2 Receptor Agonists/pharmacology ; Biosensing Techniques/*methods ; Cell Membrane/chemistry/metabolism ; Clathrin-Coated Vesicles ; Cyclic AMP/metabolism ; Endocytosis ; Endosomes/chemistry/*metabolism ; GTP-Binding Protein alpha Subunits, Gs/metabolism ; Green Fluorescent Proteins/analysis/genetics/metabolism ; HEK293 Cells ; Humans ; Isoproterenol/pharmacology ; Models, Biological ; Protein Conformation ; Receptors, Adrenergic, beta-2/*chemistry/immunology/*metabolism ; *Signal Transduction ; Single-Domain Antibodies/genetics/immunology
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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