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Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

ABSTRACT: Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its ? subunit (G?T). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the G?T helical domain (?HD) contacting the G?? subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by G?? during receptor-catalyzed nucleotide exchange.

SUBMITTER: Gao Y

PROVIDER: S-EPMC6707884 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

Gao Yang Y Hu Hongli H Ramachandran Sekar S Erickson Jon W JW Cerione Richard A RA Skiniotis Georgios G

Molecular cell 20190709 4

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low ...[more]

PMID: 31300275

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The rhodopsin-transducin complex houses two distinct rhodopsin molecules.

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Coupling efficiency of rhodopsin and transducin in bicelles.

Project description:G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides, or light. After activation, receptors interact with heterotrimeric G proteins and catalyze GDP release from the G? subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the G? protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (G(t)), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-G(t)(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to 1 week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes and can extend the range of studies that investigate properties of these complexes.

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Atomistic insights into rhodopsin activation from a dynamic model.

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A C-terminal peptide of bovine rhodopsin binds to the transducin alpha-subunit and facilitates its activation.

Project description:In order to investigate the possible roles of the intracellular domains of rhodopsin in the functional coupling of the photoreceptor to transducin, different peptides that correspond to parts of the known sequence of rhodopsin were synthesized. Since we have found tht the binding of rhodopsin to the alpha subunit of transducin (alpha T) increases the susceptibility of alpha T to phosphorylation by protein kinase C-beta 1, we used this phosphorylation reaction as an initial screen for peptides that mimic the actions of rhodopsin. The results of this screen indicated that a peptide from the C-terminal tail of rhodopsin (amino acids 325-338; KNPLGDDEASTTVS-amide; designated as peptide 3) was capable of interacting with the alpha T subunit. Evidence that peptide 3 binds to alpha T at a site that overlaps the rhodopsin-binding domain was obtained from experiments showing that peptide 3 inhibited the rhodopsin-stimulated GTPase activity of alpha T and that this inhibition was overcome at high levels of rhodopsin. A potentially important outcome of the peptide 3/alpha T interaction is the facilitation of the activation of the alpha T subunit. This was first demonstrated in fluorescence experiments where the binding of peptide 3 was shown to strongly promote the enhancement of the tryptophane emission of alpha T that is elicited by the addition of NaF. Specifically, the EC50 for NaF was shifted from approximately 4 mM in the absence of peptide 3 to below 0.5 mM in the presence of peptide 3. Further verification that peptide 3 facilitated the ability of NaF to activate the alpha T subunit was obtained from experiments measuring the alpha T GDP/NaF-stimulated hydrolysis of cyclic GMP by the cyclic GMP phosphodiesterase.

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