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Energetic analysis of the rhodopsin-G-protein complex links the ?5 helix to GDP release.

ABSTRACT: We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the ?2 adrenergic receptor-Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the G? C-terminal helix (?5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening compared with the crystal structure. The ?5 helix undergoes a 63° rotation, accompanied by a 5.7-Å translation, that reorganizes interfaces between ?5 and ?1 helices and between ?5 and ?6-?5. Changes in the ?6-?5 loop displace ?G. All of these movements lead to opening of the GDP-binding pocket. The model creates a roadmap for experimental studies of receptor-mediated G-protein activation.

SUBMITTER: Alexander NS

PROVIDER: S-EPMC3947367 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release.

Alexander Nathan S NS Preininger Anita M AM Kaya Ali I AI Stein Richard A RA Hamm Heidi E HE Meiler Jens J

Nature structural & molecular biology 20131201 1

We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the β2 adrenergic receptor-Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the Gα C-terminal helix (α5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain op ...[more]

PMID: 24292645

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Project description:BackgroundRhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear.Principal findingsWe herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4'-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108-135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees.SignificanceAccessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.

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Energetic analysis of the rhodopsin-G-protein complex links the ?5 helix to GDP release.

Publications

Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release.

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