title: Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.
abstract: In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.
authors.affiliation: From the Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402.
authors.forename: Jagjeet S
authors.initials: JS
authors.lastname: Mnpotra
authors.affiliation: the Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292.
authors.forename: Zhuanhong
authors.initials: Z
authors.lastname: Qiao
authors.affiliation: the Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292.
authors.forename: Jian
authors.initials: J
authors.lastname: Cai
authors.affiliation: From the Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402.
authors.forename: Diane L
authors.initials: DL
authors.lastname: Lynch
authors.affiliation: the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, and.
authors.forename: Alan
authors.initials: A
authors.lastname: Grossfield
authors.affiliation: the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, and.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Leioatts
authors.affiliation: From the Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402.
authors.forename: Dow P
authors.initials: DP
authors.lastname: Hurst
authors.affiliation: From the Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402, the Computational Biology Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Pitman
authors.affiliation: the Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292, zhsong@louisville.edu.
authors.forename: Zhao-Hui
authors.initials: ZH
authors.lastname: Song
authors.affiliation: From the Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, North Carolina 27402, phreggio@uncg.edu.
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Reggio
mesh_terms: D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C588309:CNR2 protein, human; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D043885:Receptor, Cannabinoid, CB2; D011994:Recombinant Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Cannabinoid Receptor; G Protein-coupled Receptor (GPCR); Mass Spectrometry (MS); Molecular Dynamics; Signal Transduction