Spatial Organization of Lipids Drives GPCR Conformational Equilibria

Despite the widespread use of lipid nanodiscs for structure-function studies of membrane proteins, little is known about how lipids are spatially organized within nanodiscs and how this organization influences embedded proteins. The activity and conformational equilibria of the human A _2A adenosine receptor, a class A G protein-coupled receptor, are highly sensitive to anionic lipids that directly interact with the receptor. We leverage this lipid-dependent sensitivity to probe the accessibility of anionic lipids across nanodiscs of varying sizes. We identify a threshold concentration of anionic lipids required to fully populate active receptor conformations that is higher for POPS than for POPG. Computational simulations reveal that POPS and POPG each form lipid clusters reducing the effective availability of anionic lipids to interact with the receptor. This effect is stronger for POPS and scales with increasing nanodisc size, correlating with experimental biophysical and biochemical measurements. Simulations further identify positively charged residues within the membrane scaffold protein that coordinate anionic lipid headgroups. Targeted protein engineering reduces the threshold concentration of anionic lipids required for receptor activation, supporting strategies to control lipid accessibility within nanodiscs. Because membrane scaffold proteins are derived from apolipoprotein A-I, similar lipid-protein interactions may also influence lipid organization within biological systems such as HDL particles.