Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction: FORMATION OF THE CANNABINOID CB

• Published in: The Journal of biological chemistry 2014-05, Vol.289 (29), p.20259-20272
• Main Authors: Mnpotra, Jagjeet S., Qiao, Zhuanhong, Cai, Jian, Lynch, Diane L., Grossfield, Alan, Leioatts, Nicholas, Hurst, Dow P., Pitman, Michael C., Song, Zhao-Hui, Reggio, Patricia H.
• Format: Article
• Language: English
• Subjects: Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.539916

Background: CB 2 couples with only G i protein. Results: Cross-linking studies using LC-MS/MS and ESI-MS/MS identified three specific CB 2 -Gα i cross-link sites. MD showed an orientation change from the β2-AR*/G s geometry makes all cross-links possible. Conclusion: Second intracellular loop of CB 2 interactions are key for G i complex formation. Significance: Findings should be relevant for other GPCRs that couple to G i proteins. In this study, we applied a comprehensive G protein-coupled receptor-Gα i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB 2 )- Gα i 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 Gα i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gα i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gα i α 4 β 6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB 2 ·G i complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB 2 R*·Gα i1 β 1 γ 2 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*·Gα s β 1 γ 2 complex crystal structure, the Gα i1 β 1 γ 2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gα i1 α5 helix tilt changed due to the outward movement of TMH5 in CB 2 R*. 2) A 25° clockwise rotation of Gα i1 β 1 γ 2 underneath CB 2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gα i1 (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 G i proteins.