Solid-State NMR Study of the Human Peripheral Cannabinoid Receptor CB2 in Lipid Bilayers

Abstract

Human peripheral cannabinoid receptor CB2 expressed in E. coli. has been purified, and successfully reconstituted in functional form into lipid bilayers composed of phosphatidylcholine, phosphatidylserine, and cholesteryl hemisuccinate (CHS). The reconstitution was carried out by detergent removal from the CB2-lipid-detergent mixed micelles on an adsorbent column, or by rapid dilution below the critical micelle concentration followed by washing on a concentrator. Proteoliposomes prepared at the CB2:phospholipid molar ratio of 1:600 showed the following basic physical properties: Free of detergents (as analyzed by high-resolution 1H NMR), homogeneity of the CB2-to-lipid ratio over the proteoliposome particles (by sucrose gradient centrifugation), unilamellarity with a mean diameter of ∼150-200 nm (by dynamic light scattering), and functional integrity (by G-protein activation assay). Using the proteoliposomes, CB2-lipid interactions were investigated by solid-state NMR. Order parameters of the phospholipid acyl chains measured by 2H quadrupolar splittings indicated that CHS, a detergent-soluble analog of cholesterol, preferentially interacts with the 1-palmitoyl-2-oleoyl-sn-glycero-3-phospholipids over CB2. By probing 1H NMR saturation transfer, evidence for CB2-lipid interactions at the lipid acyl chains and less significant interactions at the glycerol backbone and the headgroups were observed. 1H spin-lattice relaxation rates decreased notably at the acyl-chains upon CB2 incorporation, indicating reduced motion on the nanosecond timescale corresponding to the restriction of phospholipid wobbling about the bilayer normal. Structure-function relationships in view of the role of interactions between CB2 and anionic phosphatidylserine in activation of G-protein will also be discussed.