Ric-8a Catalyzed G Protein Activation Proceeds Through a Disordered State

Abstract

Members of the Ras superfamily of regulatory GTP binding proteins, Heterotrimeric G protein alpha subunits (Ga) undergo cycles of activation and deactivation driven by binding and hydrolysis of GTP. Activation occurs by replacement of GDP by GTP at the nucleotide binding site of Ga, which requires catalytic assistance from guanine nucleotide exchange factors (GEFs). Transmembrane G protein-coupled Receptors (GPCRs) are the best known G protein GEFs, but recently, a novel family of cytosolic, non-receptor GEFs, typified by mammalian Ric-8A, were discovered. Unlike GPCRs, which act on G protein heterotrimers, Ric-8A catalyzes the release of GDP directly upon Gi-class Ga subunits (Gai), and has negligible affinity for Gai-GTP. Upon binding to Gai-GDP, Ric-8A catalyzes GDP release and forms a stable Gai:Ric-8A complex that dissociates only in the presence of GTP, resulting in the release of Gai-GTP. The TROSY-HSQC spectrum of [1H,15N]Gai bound to Ric-8A is considerably broadened relative to Gai-GDP. Hydrogen-deuterium exchange mass spectroscopy shows that the rate of HD exchange at Gai:Ric-8A is more than 2X faster than from Gai-GDP. Differential scanning calorimetry shows that both Ric-8A and Gai-GDP undergo cooperative, irreversible unfolding transitions at 47 deg and 52 deg, respectively, while nucleotide-free Gai shows a broad, weak transition near 35 deg. The unfolding transition for Gai:Ric-8A is complex, with a broad transition peaking at 49i. Ric-8A therefore stabilizes nucleotide-free Gai in a dynamic state, which, we propose, facilitates GTP binding. We show that the C-terminus of Gai is a critical binding element for Ric-8A, as is known to be the case for GPCRs, suggesting that these two GEFs act by similar mechanisms as chaperones for the unstable and dynamic nucleotide-free state of Ga.