Heterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits.

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TitleHeterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits.
Publication TypeJournal Article
Year of Publication2005
AuthorsAbdulaev, NG, Ngo, T, Zhang, C, Dinh, A, Brabazon, DM, Ridge, KD, Marino, JP
JournalJ Biol Chem
Date Published2005 Nov 11
KeywordsEscherichia coli, Gene Expression, GTP-Binding Protein alpha Subunits, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Magnesium, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Conformation

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein betagamma-subunit (G(betagamma)) and activated receptor (R(*)) interactions. Here, we show that ChiT can be functionally reconstituted with G(betagamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that (15)N-ChiT can be titrated with G(betagamma) to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the (15)N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg(2+)-bound (15)N-ChiT in the presence and absence of aluminum fluoride and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)/Mg(2+)-bound (15)N-ChiT. As anticipated, G(betagamma) association with (15)N-ChiT results in (1)HN, (15)N chemical shift changes relative to the GDP/Mg(2+)-bound state. Strikingly, however, most (1)HN, (15)N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP.AlF(4)(-)/Mg(2+)- and GTPgammaS/Mg(2+)-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of G(alpha) ("preactivation") that may facilitate the interaction with R(*) and subsequent GDP/GTP exchange.

Alternate JournalJ. Biol. Chem.
PubMed ID16129667
Grant ListRR015744 / RR / NCRR NIH HHS / United States