Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

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 α-subunit (G α ) chimera (ChiT) associated with G-protein βγ-subunit (G βγ ) and activated receptor (R * ) interactions. Here, we show that ChiT can be functionally reconstituted with G βγ 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 βγ 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 (GTPγS)/Mg 2+ -bound 15 N-ChiT. As anticipated, G βγ 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 - and GTPγS/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 α (“preactivation”) that may facilitate the interaction with R * and subsequent GDP/GTP exchange.