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Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Molecular basis for high-affinity agonist binding in GPCRs Tony Warne , Patricia C. Edwards , Andrew Dore , Andrew G. W. Leslie , Christopher G. Tate Science DOI: 10.1126/science.aau5595 Diamond Proposal Number(s): [8547] Show Abstract ▼ 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.	May 2019
I24-Microfocus Macromolecular Crystallography	Structure of the adenosine A2A receptor bound to an engineered G protein Byron Carpenter , Rony Nehmé , Tony Warne , Andrew G. W. Leslie , Christopher G. Tate Nature 536, 104 - 107 DOI: 10.1038/nature18966 Diamond Proposal Number(s): [11235] Show Abstract ▼ 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.	Jul 2016
I24-Microfocus Macromolecular Crystallography	Pharmacological analysis and structure determination of 7-methylcyanopindolol–bound β1-adrenergic receptor T. Sato , J. Baker , Tony Warne , G. A. Brown , Andrew Leslie , M. Congreve , Chris Tate Molecular Pharmacology 88 (6), 1024 - 1034 DOI: 10.1124/mol.115.101030 PMID: 26385885 Show Abstract ▼ 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.	Oct 2015
I24-Microfocus Macromolecular Crystallography	Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening Fabrice Gorrec , Colin M. Palmer , Gui Lebon , Tony Warne Acta Crystallographica Section D Biological Crystallography 67 (5), 463 - 470 DOI: 10.1107/S0907444911008754 PMID: 21543849 Open Access Show Abstract ▼ Abstract: The Pi sampling method is derived from the incomplete factorial approach to macromolecular crystallization screen design. The resulting `Pi screens' have a modular distribution of a given set of up to 36 stock solutions. Maximally diverse conditions can be produced by taking into account the properties of the chemicals used in the formulation and the concentrations of the corresponding solutions. The Pi sampling method has been implemented in a web-based application that generates screen formulations and recipes. It is particularly adapted to screens consisting of 96 different conditions. The flexibility and efficiency of Pi sampling is demonstrated by the crystallization of soluble proteins and of an integral membrane-protein sample.	May 2011

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