The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic.

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TitleThe receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic.
Publication TypeJournal Article
Year of Publication2006
AuthorsAbdulaev, NG, Ngo, T, Ramon, E, Brabazon, DM, Marino, JP, Ridge, KD
Date Published2006 Oct 31
KeywordsAnimals, Cattle, GTP-Binding Protein alpha Subunits, Guanine Nucleotides, Guanosine Diphosphate, Guanosine Triphosphate, Heterotrimeric GTP-Binding Proteins, Light, Magnetic Resonance Spectroscopy, Models, Biological, Models, Chemical, Models, Molecular, Protein Binding, Protein Conformation, Receptors, G-Protein-Coupled, Rhodopsin, Transducin

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.

Alternate JournalBiochemistry
PubMed ID17059215
Grant ListEY013286 / EY / NEI NIH HHS / United States
EY016493 / EY / NEI NIH HHS / United States
RR015744 / RR / NCRR NIH HHS / United States