G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain14. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AAR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure1 and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure2, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors

Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F6.52. Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for drug discovery.N.F. acknowledges support from Generalitat de Catalunya (FI-Agaur). GDF acknowledges support from MINECO (BIO2014-53095-P) and FEDER. We also thank all the volunteers of GPUGRID who donated GPU computing time to the project

Structure of a D2 dopamine receptor–G-protein complex in a lipid membrane

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease(1)and antipsychotic drugs(2). DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine(3), leading to stimulation of G(i)and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-G(i)complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular G(i)-binding site. The DRD2-G(i)structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.The structure of the D2 dopamine receptor in complex with its G protein reveals how dopamine receptors are activated and, importantly, how a G-protein-coupled receptor can interact with its G protein in a phospholipid membrane