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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

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Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

Mnpotra Jagjeet S JS Qiao Zhuanhong Z Cai Jian J Lynch Diane L DL Grossfield Alan A Leioatts Nicholas N Hurst Dow P DP Pitman Michael C MC Song Zhao-Hui ZH Reggio Patricia H PH

The Journal of biological chemistry 20140522 29

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 ...[more]

PMID: 24855641

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Project description:Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.

Dataset Information

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

Publications

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

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