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-G?i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-G?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 G?i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the G?i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the G?i ?4?6 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*·G?i1?1?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 ?2-AR*·G?s?1?2 complex crystal structure, the G?i1?1?2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The G?i1 ?5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of G?i1?1?2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on G?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 Gi proteins.

SUBMITTER: Mnpotra JS 

PROVIDER: S-EPMC4106340 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature