Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Abstract: A third of all FDA approved drugs target G protein-coupled receptors (GPCRs) but often have side effects. GPCRs have two signalling pathways - the G protein-coupled pathway and the arrestin-mediated pathway - and one may be the therapeutic pathway whilst the other may produce side effects. To understand this phenomenon at the molecular level, researchers needed to determine the structure of a GPCR coupled to arrestin and compare its structure to the G protein-coupled state, with the same agonist bound to both receptors. The GPCR-arrestin complex is highly mobile, which means it would not form crystals suitable for X-ray diffraction experiments. Therefore, the team used cryogenic-electron microscopy (cryo-EM) at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source to determine its structure at a 3.3Å resolution.
Producing a stable GPCR-arrestin complex suitable for structure determination was still a challenge. Arrestin coupling to a GPCR requires the presence of membrane lipids, so they prepared the receptor in lipid nanodiscs. The receptor also has to be phosphorylated, so they developed a method for ligating phosphorylated peptides to the end of the receptor. Their results allowed them to identify two regions of the GPCR that could be used in the development of ‘biased’ agonists that signal predominantly through either the G protein or arrestin pathways. The structures will be an invaluable input into structure-based drug design, a powerful tool for the development of new therapeutic drugs.
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Jul 2021
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Krios I-Titan Krios I at Diamond
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Yang
Lee
,
Tony
Warne
,
Rony
Nehmé
,
Shubhi
Pandey
,
Hemlata
Dwivedi-Agnihotri
,
Madhu
Chaturvedi
,
Patricia C.
Edwards
,
Javier
Garcia-Nafria
,
Andrew G. W.
Leslie
,
Arun K.
Shukla
,
Christopher G.
Tate
Diamond Proposal Number(s):
[17434]
Abstract: The β1-adrenoceptor (β1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of β-arrestin 1 (βarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins—known as biased agonism3—is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the β1AR–βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound β1AR coupled to the G-protein-mimetic nanobody6 Nb80. βarr1 couples to β1AR in a manner distinct to that7 of Gs coupling to β2AR—the finger loop of βarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in βarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. β1AR coupled to βarr1 shows considerable differences in structure compared with β1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of β1AR, and find that formoterol has a lower affinity for the β1AR–βarr1 complex than for the β1AR–Gs complex. The structural differences between these complexes of β1AR provide a foundation for the design of small molecules that could bias signalling in the β-adrenoceptors.
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Jun 2020
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8547]
Abstract: G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists compared to when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the β1-adrenoceptor (β1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β1AR bound to the identical ligands showed a 24-42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.
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May 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11235]
Abstract: G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary1, 2. Here we present the structure of the adenosine A2A receptor (A2AR) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A2AR through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the β2-adrenergic receptor3. The transition of the receptor from an agonist-bound active-intermediate state4, 5 to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A2AR–mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs6 and hints at the potential complexity of the molecular basis for G-protein specificity.
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Jul 2016
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I24-Microfocus Macromolecular Crystallography
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Abstract: Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser5.46, coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol–bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol–bound β1AR and the hydroxyl group of Ser5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser5.46 side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.
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Oct 2015
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I24-Microfocus Macromolecular Crystallography
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Alexander
Heifetz
,
Gebhard F. X.
Schertler
,
Roland
Seifert
,
Christopher G.
Tate
,
Patrick M.
Sexton
,
Vsevolod V.
Gurevich
,
Daniel
Fourmy
,
Vadim
Cherezov
,
Fiona H.
Marshall
,
R. Ian
Storer
,
Isabel
Moraes
,
Irina G.
Tikhonova
,
Christofer S.
Tautermann
,
Peter
Hunt
,
Tom
Ceska
,
Simon
Hodgson
,
Mike J.
Bodkin
,
Shweta
Singh
,
Richard J.
Law
,
Philip C.
Biggin
Diamond Proposal Number(s):
[5953, 11386]
Open Access
Abstract: G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future.
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Mar 2015
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I24-Microfocus Macromolecular Crystallography
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John
Christopher
,
Jason
Brown
,
Andrew
Dore
,
James
Errey
,
Markus
Koglin
,
Fiona H.
Marshall
,
Dave
Myszka
,
Rebecca L.
Rich
,
Christopher G.
Tate
,
Benjamin
Tehan
,
Tony
Warne
,
Miles
Congreve
Open Access
Abstract: Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein–ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.
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Mar 2013
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I24-Microfocus Macromolecular Crystallography
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Robin
Owen
,
Danny
Axford
,
Joanne E.
Nettleship
,
Raymond J.
Owens
,
James I.
Robinson
,
Ann W.
Morgan
,
Andrew S.
Dore
,
Guillaume
Lebon
,
Christopher G.
Tate
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
,
Gwyndaf
Evans
Open Access
Abstract: A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin [gamma] Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.
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Jul 2012
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6641]
Open Access
Abstract: The β1-adrenoceptor (β1AR) is the site of action of beta blockers used in the treatment of cardiac-related illnesses. Two beta blockers, carvedilol and bucindolol, show distinctive activities compared to other beta blockers and have been proposed as treatments tailored to the Arg/Gly3898.56 polymorphism of the human β1AR. Both carvedilol and bucindolol are classified as biased agonists, because they stimulate G protein-independent signaling, while acting as either inverse or partial agonists of the G protein pathway. We have determined the crystal structures of a thermostabilized avian β1AR mutant bound to bucindolol and to carvedilol at 3.2 and 2.3 Å resolution, respectively. In comparison to other beta blockers, bucindolol and carvedilol interact with additional residues, in extracellular loop 2 and transmembrane helix 7, which may promote G protein-independent signaling. The structures also suggest that there may be a structural explanation for the pharmacological differences arising from the Arg/Gly3898.56 polymorphism.
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May 2012
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I24-Microfocus Macromolecular Crystallography
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Andrew S.
Dore
,
Nathan
Robertson
,
James C.
Errey
,
Irene
Ng
,
Kaspar
Hollenstein
,
Ben
Tehan
,
Edward
Hurrell
,
Kirstie
Bennett
,
Miles
Congreve
,
Francesca
Magnani
,
Christopher G.
Tate
,
Malcolm
Weir
,
Fiona H.
Marshall
Diamond Proposal Number(s):
[6641]
Abstract: Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.
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Sep 2011
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