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 alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein beta gamma-subunit (G(beta gamma)) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with G(beta gamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that N-15-ChiT can be titrated with G(beta gamma) to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the N-15-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound N-15-ChiT in the presence and absence of aluminum fluoride and guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)/ Mg2+-bound N-15-ChiT. As anticipated, G(beta gamma) association with N-15-ChiT results in (HN)-H-1, N-15 chemical shift changes relative to the GDP/ Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP center dot AlF4-/Mg2+- and GTP gamma S/Mg2+-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.