Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 A X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

/pdf/structural-insights-into-u-opioid-receptor-activation-s771o46rw6.pdf

Structural insights into µ-opioid receptor activation

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

/pdf/the-molecular-basis-of-g-protein-coupled-receptor-activation-4pv2iekf2l.pdf

The Molecular Basis of G Protein-Coupled Receptor Activation.

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(14)00048-6.pdf

Allosteric sodium in class A GPCR signaling

An engineered G protein is used to bind to and stabilize the active conformation of the adenosine A2A receptor, enabling the acquisition of an X-ray crystal structure of this GPCR in an active state. G-protein-coupled receptors (GPCRs) are essential components of signalling networks throughout the body, and about a third of all clinical drugs target GPCRs. The X-ray structures of GPCRs in an active conformation have proved elusive. This paper describes the crystal structure of adenosine A2A receptor bound to a G protein, which represents the first X-ray structure of the fully active state of the receptor. The trick used here involved engineering a G protein — termed mini-Gs — that binds to and stabilizes the active state of the adenosine A2A receptor. The hope is that this mini-Gs will facilitate the crystallization and characterization of other Gs-coupled GPCRs in their active states. 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 A resolution. Mini-Gs binds to A2AR through an extensive interface (1,048 A2) 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 A 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.

/pdf/structure-of-the-adenosine-a2a-receptor-bound-to-an-32cw4jh0k6.pdf

Structure of the adenosine A2A receptor bound to an engineered G protein

G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics.

Structural insights into G-protein-coupled receptor allostery.

The β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 A resolution. High-quality crystals (100 μm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 A), despite an overall rmsd of 2.4 A for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5–10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.

/pdf/the-2-1-a-resolution-structure-of-cyanopindolol-bound-b1-wv3q7bqrns.pdf

The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online) 

Jennifer L. Miller-Gallacher

Papers

The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies

Cryo-electron microscopy structure of full length TSH receptor in complex with TSH receptor blocking human monoclonal autoantibody K1-70TM