Probing the molecular mechanism of heterotrimeric G‐protein signaling using high‐resolution NMR

Abstract

The structural basis for activation of heterotrimeric (α β γ) G-proteins by G-protein coupled receptors (GPCRs) is not well understood. Using the rhodopsin/transducin system, conformational changes in Gα accompanying heterotrimer formation and activated GPCR (R∗) catalyzed GDP/GTP exchange are being tracked by high-resolution NMR. We have shown that an 15N-labeled Gα chimera (ChiT) that displays relatively well-dispersed 2D spectra and uniform line widths undergoes aluminum fluoride induced perturbations in switch II and at the C-terminus; some anticipated R∗- and GTP-induced perturbations in these same regions are already evident upon heterotrimer formation, likely providing kinetic advantages in R∗/G-protein coupling; R∗- and Gβ γ-released exchanged ChiT displays further C-terminal perturbation and increased conformational flexibility of switch II, which may be important for Gα/effector interactions and GTP hydrolysis; the GDP-released, R∗-bound state of ChiT shows severe line broadening suggestive of a dynamic intermediate that results from changes in the R∗-interacting N- and C-termini; and N- and C-terminal mutations can have long-range consequences on ChiT conformation, particularly in switch II. These results provide new insights into the mechanism of signal propagation from R∗ to the G-protein. Support by NIH, Robert A. Welch Foundation, and Spanish Ministry of Science.