Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein a subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Ga Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gai1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Ga interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Ga. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Ga and possibly at the Ga domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.