Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

Abstract

The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and G s protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock G s protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.

Statistics for the analysis of molecular dynamics simulations: providing P values for agonist-dependent GPCR activation

“…In the present article we reanalyze a recent study of ours in which the activation of the adenosine A2a Class A GPCR (A2aR) was examined 7 . In this study, 2 µs-length MD simulations of A2aR under 2 experimental conditions: absence/presence of endogenous adenosine and DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine)/ DOPG (1,2-dioleoyl-sn-glycerol-3-phosphoglycerol) lipid environment were performed 7 . To provide sufficient variability, 4 replicas were run in each condition value.…”

“…As in the case of adenosine, these trajectories are described individually in Ref. 7 . Considering that NECA is a The TM3-TM6 distance is measured between Cα atoms of R102 3.50 and E288 6.30 .…”

“…As in the case of adenosine, these trajectories are described individually in Ref. 7 . Considering that NECA is a more potent agonist than adenosine one would expect that the performed statistics would reflect this pharmacological feature.…”

“…Horizontal TM3–TM6 and TM3–TM7 red lines correspond to the distances between the aforementioned respective atoms for the inactive receptor (PDB entry: 4EIY) 16 whereas horizontal green lines correspond to the distances for the active receptor (PDB entry: 6GDG) 17 . Figures adapted from Ref 7 .

Figure 4 Descriptive statistics, mean and standard error of the mean (SEM), for the sampled 4 replicas in each of the conditions (lipid, NECA) depicted in Fig.

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In Silico Drug Design for Purinergic GPCRs: Overview on Molecular Dynamics Applied to Adenosine and P2Y Receptors

“…Lipid allostery on A 2A AR contributes to an active-like conformation, as shown using unbiased, long (µsec) MD simulations [124,125]. In addition, receptor-cholesterol interactions have been explored with MD simulations [126][127][128], including the observation of cholesterol entering the receptor, going beyond its solely allosteric effect [129].…”