Two-Dimensional NMR Spectroscopy of the G Protein-Coupled Receptor A 2A AR in Lipid Nanodiscs.

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure-function relationships and thus benefits continued drug development. Here, we incorporate the human A 2A adenosine receptor (A 2A AR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [ 15 N, 1 H]-TROSY correlation spectra confirmed that the complex of [u- 15 N, ~70% 2 H]-A 2A AR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A 2A AR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A 2A AR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15 N- 1 H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A 2A AR. As expected, the largest effects were observed near the lipid-water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.