Insights into adenosine A2Areceptor activation through cooperative modulation of agonist and allosteric lipid interactions.

Agustín Bruzzese,James A R Dalton,Jesús Giraldo,Bert L De Groot

doi:10.1371/journal.pcbi.1007818

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 Gs 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 Gs 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.

Highlights

G protein-coupled receptors (GPCRs) are heptahelical transmembrane proteins [1, 2]
Unbiased microsecond-length Molecular Dynamics (MD) simulations of the adenosine A2a receptor (A2aR) were performed in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: adenosine or NECA, each of them embedded in two different homogeneous phospholipid membranes
Dynamic structural data are key for the understanding of agonist-mediated GPCR activation simulated in realistic membrane environments

Summary

Introduction

G protein-coupled receptors (GPCRs) are heptahelical transmembrane proteins [1, 2]. In the simplest scheme, they mediate many physiological and pathological processes by transduction of signals across cellular membranes and exist in a conformational equilibrium between active and inactive forms [3, 4]. In the active state are GPCRs able to bind cytosolic proteins, such as G protein or β-arrestin, mediating different downstream signalling pathways from the same receptor [9, 10]. Ligand binding triggers conformational changes in the orthosteric site that are amplified into larger conformational rearrangements [6]. For these reasons, GPCRs are one of the major targets of current market drugs [13]. It is essential to deeply understand ligand-dependent (de)activation of GPCRs in order to suitably understand human physiology and expectedly perform more efficient drug discovery

Methods

Results

Discussion

Conclusion