The receptor-bound empty pocket state of the heterotrimeric G-protein α-subunit is conformationally dynamic

Heterotrimeric G-protein activation by a G-protein-coupled receptor ( GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G alpha) associated with activated GPCR ( R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled GR reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [ Ridge, K. D., et al. ( 2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R* form trapped intermediate complexes with a GDP-released '' empty pocket '' state of the heterotrimer in the absence of GTP ( or GTP-S). In contrast to guanine nucleotide-bound forms of GR, the NMR spectra of the GDP-released, R*-bound empty pocket state of GR display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg2+-bound state of GR generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg2+-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of GR, and not the absence of bound guanine nucleotide, are the predominant factors which dictate GR conformation and dynamics in this R*-bound state of the heterotrimer.