G-protein coupled receptors (GPCR) in Membrane Bilayers
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulating immune responses, was prepared and immobilized on a monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was shown by surface plasmon resonance (SPR) to be captured quantitatively on 1D4-coated CM4 chips. Locatelli-Hoops SC, Gorshkova I, Gawrisch K, Yeliseev AA. Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2. Biochim Biophys Acta. 2013 Oct;1834(10):2045-56. doi: 10.1016/j.bbapap.2013.06.003.
Human cannabinoid type 2 (CB2) receptor was purified and reconstituted in a functional form into lipid bilayers composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesteryl hemisuccinate (CHS). The efficiency of G-protein activation by agonist-bound CB2 receptor was affected by negative electric surface potentials of proteoliposomes controlled by the content of anionic CHS or POPS. The activation was highest at an anionic lipid content of about 50 mol %. Kimura T, Yeliseev AA, Vukoti K, Rhodes SD, Cheng K, Rice KC, Gawrisch K. Recombinant cannabinoid type 2 receptor in liposome model activates G-protein in response to anionic lipid constituents. J Biol Chem. 2012 Feb 3;287(6):4076-87. doi: 10.1074/jbc.M111.268425.
To test the hypothesis that endogenous cannabinoid sn-2-arachidonoylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer, we employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) 2-AG first partitions out of bulk lipid at the transmembrane alpha-helix (TMH) 6/7 interface; 2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the 2-AG headgroup into the CB2 binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. Hurst DP, Grossfield A, Lynch DL, Feller S, Romo TD, Gawrisch K, Pitman MC, Reggio PH. A lipid pathway for ligand binding is necessary for a cannabinoid G protein-coupled receptor. J Biol Chem. 2010 Jun 4;285(23):17954-64. doi: 10.1074/jbc.M109.041590.
Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatography followed by ion-exchange chromatography. By high-resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques. Yeliseev AA, Wong KK, Soubias O, Gawrisch K. Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci. 2005 Oct;14(10):2638-53.
A quantitative analysis of lipid-order parameter changes suggested that the rhodopsin protein adjusts its conformation to bilayer hydrophobic thickness. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. Increasing bilayer thickness favors formation of photointermediate metarhodopsin II, whereas oligomerization favors photointermediate metarhodopsin I. Soubias O, Teague WE Jr, Hines KG, Gawrisch K. Rhodopsin/lipid hydrophobic matching-rhodopsin oligomerization and function. Biophys J. 2015 Mar 10;108(5):1125-32. doi: 10.1016/j.bpj.2015.01.006.
Docosahexaenoic acid chain (DHA, 22:6n-3) interactions with rhodopsin have far more rapid structural conversions than saturated and monounsaturated hydrocarbon chains. Furthermore, DHA chains tend to have higher density near the lipid/water interface while density of saturated chains is higher in the bilayer center. Also, polyunsaturated phosphatidylethanolamines accumulate preferentially near the protein. Surprisingly, the high conformational freedom of most DHA chains is not measurably reduced upon interaction with rhodopsin. Gawrisch K, Soubias O, Mihailescu M. Insights from biophysical studies on the role of polyunsaturated fatty acids for function of G-protein coupled membrane receptors. Prostaglandins Leukot Essent Fatty Acids. 2008 Sep-Nov;79(3-5):131-4.
Specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. The molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins. Koenig BW, Gawrisch K. Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase. Biochim Biophys Acta. 2005 Aug 30;1715(1):65-70.
updated October, 2017