Showing papers by "Andrew G. W. Leslie published in 2016"

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

Abstract: 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.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.

Abstract: The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the e-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the e-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the e-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated e-subunit unable to bind ATP, the enzyme remains inactive and the e-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the e-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.