In the present study, we applied a comprehensive GPCR- Gαi, protein chemical cross-linking strategy to map the cannabinoidreceptor subtype 2 (CB2) – Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three crosslink sites were identified using LC MS/MS and ESI MS/MS (1) a sulfhydryl crosslink between C3.53(134) in TMH3 and the Gαi, C-terminal i-3 residue, C351; (2) a lysine crosslink between K6.35(245) in TMH6 and the Gαi, C-terminal i-5 residue, K349; and (3) a lysine crosslink between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, K317. To investigate the dynamics and nature of the conformational changes involved in CB2-Gi complex formation, we carried out microsecond-timescale molecular dynamics (MD) simulations of the CB2 R*/ Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer, using crosslinking information as validation. Our results show that although MD simulations started with the G protein orientation in the β2-AR*/ Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein re-oriented itself within 300 ns. Two major changes occurred: (1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. (2) A 25 degree clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when P139 (IC2 loop) anchors in a hydrophobic pocket on Gαi1 (V34, L194, F196, F336, T340, I343 and I344). In this complex, all three experimentally identified crosslinks can occur. These findings should be relevant for other Class A GPCRs that couple to Gi proteins.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

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