Activation of the A2A adenosine G-protein-coupled receptor by conformational selection

Abstract

Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

Conformational equilibria of light-activated rhodopsin in nanodiscs

“…In the β 2 adrenergic receptor and the adenosine A 2A receptor, multiple conformations were found in both the unliganded protein and with an inverse agonist bound, also suggesting a weak coupling of the cytoplasmic surface with the ligand-binding site (2,24).…”

“…Collectively, the data from SDSL-EPR in DDM and crystal structures give the impression that rhodopsin is unique among GPCRs, acting essentially as a binary switch, moving from a unique inactive conformation to one representing the activated state. This would be in contrast to other GPCRs, which apparently have a complex conformational landscape even in detergent solutions (2,24). One goal of this study is to directly examine the conformational repertoire of rhodopsin in nativelike lipid bilayers using SDSL-EPR spectroscopy.…”

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

“…A 2A R (Ye et al, 2016) provided evidence for at least four different conformational states populated by these receptors, which are differentially stabilized by ligands of different efficacy.…”

“…Within the slow millisecond-timescale global interconversion between functional GPCR conformations (Vilardaga et al, 2003), local structural rearrangements of sidechains in the receptor (referred to here as 'microswitches') have been observed in comparisons of inactive and active GPCR crystal structures (Katritch et al, 2013), NMR data (Liu et al, 2012a;Manglik et al, 2015;Ye et al, 2016;Bokoch et al, 2010) and MD simulations (Vanni et al, 2009;Dror et al, 2011a). The importance of repacking of sidechains from TM3, TM5, and TM6 beneath the ligand binding pocket, known as the 'conserved core triad,' was identified by comparison of structures of b 2 AR (Rosenbaum et al, 2011;Rasmussen et al, 2011a) and the m-opioid receptor .…”

Cracking the allosteric code of NMR chemical shifts

“…1). Falk et al (2) show that not all residues of TS exhibit chemical shift signatures consistent with the MWC, KNF, or the simplest EAM models, suggesting the need for more complex schemes that go beyond these allosteric paradigms (20). In this respect, we anticipate that the work by Falk et al (2) may inform further theoretical development, especially in the context of more general EAMs developed by Hilser and coworkers (19).…”