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

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

“…To improve the efficiency of production of mature, folded receptors, we made several modifications to the MFa sequence, based on biochemical and genetic precedents (Rakestraw et al, 2009;Lin-Cereghino et al, 2013) for improved processing efficiency ( Figure 1A, Materials and methods). was 0.42 nM in NaCl-containing buffer, while the K i for NECA was 210 nM in low ionic strength and 420 nM in KCl-containing buffer, in agreement with previous binding studies (Xu et al, 2011;Carpenter et al, 2016;Bertheleme et al, 2013) We developed a purification protocol for A 2A R from Pichia-derived membranes, in which we can solubilize and purify~0.5 mg of monodisperse biochemically pure wild-type A 2A R in dodecylmaltoside (DDM) detergent from 1 L of culture (Figure 1-figure supplement 1). Coupling this protocol with our previously-described labeling method (Clark et al, 2015) Figure 1B).…”

“…The Na + site first seen in the high-resolution structure of A 2A R bound to ZM241285 is conserved throughout most Class A GPCRs, and Na + exerts a negative allosteric effect on A 2A R activation by bridging residues on TM3 and TM7 and stabilizing the inactive conformation (Liu et al, 2012b). This effect can be observed pharmacologically as a NaCl-dependent decrease in agonist affinity (Carpenter et al, 2016;Gao and Ijzerman, 2000). For A 2A R expressed in P. pastoris, we observe a 9-fold decrease in NECA affinity when membranes are incubated in 150 mM NaCl buffer versus 150 mM KCl buffer (Figure 1-figure supplement 2), similar to previously reported values (Carpenter et al, 2016).…”

“…than receptor/ligand complexes ( Figure 2D), consistent with the increase in molecular weight of the complex. We also note the appearance of a few distinct peaks (most prominently at 0.85 ppm 1 H, 11.8 ppm 13 C) in the G protein complex, consistent with the conformation of the complexed receptor being distinguishable from the apo receptor (Rasmussen et al, 2011b;Carpenter et al, 2016). Assignment of 1 H/ 13 C peaks in the Ile d1 region by NMR was hampered by the modest dispersion of our spectra and the limited stability of A 2A R during data acquisition.…”

“…Here we apply this labeling method to the wild-type human A 2A R, an important regulator of vascular function with a well-developed pharmacology including the natural hormone adenosine and high-affinity synthetic agonists and antagonists (Preti et al, 2015;de Lera Ruiz et al, 2014). A 2A R has been crystallized in inactive (Jaakola et al, 2008;Liu et al, 2012b), intermediate (Lebon et al, 2011;Xu et al, 2011) and active (Carpenter et al, 2016) conformations, and has been studied by 19 F NMR to characterize its conformational equilibria (Ye et al, 2016). We expressed and purified milligram quantities of labeled wild-type human A 2A R and were able to resolve 20 out of 29 expected peaks in the Ile d1 region in 1 H/ 13 C TROSY HMQC experiments.…”

Coding GPCR-G protein specificity

“…They then filter against Gα paralogs to identify residues that may provide specificity for GPCR binding. Lastly, the selectivity residues of Gαs are mapped onto the 3 GPCR active structures that were available at the time when this manuscript was prepared (Gt peptide-bound Rhodopsin [4], mini Gαs A2A [5] and Gαsβγ:β2AR [6]), with particular emphasis on the Gαsβγ:β2AR structure. They use this interaction data to develop a footprint for GPCR residues that they propose to be involved in G protein binding selectivity, and this selectivity is informed by sequence comparison across GPCRs using the known coupling preference (IUPHAR) and the evolutionary history (e.g., comparing closely related GPCRs that have changed Gα coupling specificity).…”