Gas slow conformational transition upon GTP binding and a novel Gas regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogendeuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a ki-netic model and atomic-level characterization of stepwise conformational changes incurred by the beta(2)-adrenergic receptor (beta(2)AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gas from beta(2)AR and G(beta gamma), as opposed to a slow closing of the G(alpha s) alpha-helical domain (AHD). Yeast-two-hybrid screening using G(alpha s) AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the G(alpha s) AHD.