Heterotrimeric G-protein Α-subunit Adopts a ‘pre-activated’ 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 (Gα) chimera (ChiT) associated with G-protein βγ-subunit (Gβγ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gβγ as assessed by aluminum fluoride dependent changes in intrinsic tryptophan fluorescence, and lightactivated rhodopsin catalyzed guanine nucleotide exchange. We further show that N-ChiT can be titrated with Gβγ to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the N-ChiT reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg-bound N-ChiT in the presence and absence of aluminum fluoride, and GTPγS/Mg-bound N-ChiT. As anticipated, Gβγ association with N-ChiT results in HN, N chemical shift changes relative to the GDP/Mgbound state. Strikingly, however, most HN, N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP•AlF4/Mgand GTPγS/Mg-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 Gα (‘preactivation’) that may facilitate the interaction with R* and subsequent GDP/GTP exchange.