Solid-State NMR Study of Ligand Binding to Human Peripheral Cannabinoid Receptor CB2

Abstract

The peripheral cannabinoid receptor CB2 belongs to the family of G protein coupled receptors (GPCRs). Ligand binding studies on CB2 are complicated by high affinity of the endogenous or exogenous cannabinoid ligands for the lipid matrix that hosts the GPCR. Here we show that solid-state NMR distinguishes between specific ligand-binding to CB2 and nonspecific interactions with the lipid matrix. Experiments were conducted with recombinant, human CB2 expressed in E. coli, purified and functionally reconstituted into unilamellar liposomes. Location, structure, and dynamics of ligands in the lipid matrix were probed by NMR as well as neutron diffraction. The synthetic agonist CP-55,940 locates near the hydrophobic/hydrophilic interface of bilayers with its bond linking hydroxyphenyl and hydroxycyclohexyl rings perpendicular to the bilayer normal, while the endogenous agonist 2-AG orients parallel to the bilayer normal with the glycerol moiety near the hydrophobic/hydrophilic interface and the arachidonoyl chain in the hydrophobic region. Both ligands maintain a high level of conformational flexibility and have lateral diffusion rates in membranes comparable to those of lipids. Ligand binding to CB2 drastically shortens transverse relaxation times of the 2H-labeled ligands which distinguish between specific and non-specific binding events. Competition binding experiments with protonated and selectively deuterated CP-55,940 showed that ∼90% of the reconstituted CB2 was ligand-binding competent and formed a one-to-one complex with the ligand. Activation of G protein by agonist-bound CB2 was confirmed in a G protein activation assay. The endogenous 2-AG has a binding affinity to CB2 that is orders of magnitude lower compared to CP-55,940. The possibility that cannabinoid ligands approach the receptor from the lipid matrix will be discussed. Experiments are underway to gain structural insights into specific binding interactions based on selective isotope-labeling of both ligand and recombinant CB2.