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

• Published in: The Journal of biological chemistry 2005-11, Vol.280 (45), p.38071-38080
• Main Authors: Abdulaev, Najmoutin G, Ngo, Tony, Zhang, Cheng, Dinh, Andy, Brabazon, Danielle M, Ridge, Kevin D, Marino, John P
• Format: Article
• Language: English
• Subjects: Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; GTP-Binding Protein alpha Subunits - chemistry; GTP-Binding Protein alpha Subunits - genetics; GTP-Binding Protein alpha Subunits - metabolism; GTP-Binding Protein beta Subunits - chemistry; GTP-Binding Protein beta Subunits - genetics; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - chemistry; GTP-Binding Protein gamma Subunits - genetics; GTP-Binding Protein gamma Subunits - metabolism; Magnesium; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation
• ISSN: 0021-9258

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.