title: Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα.
abstract: Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 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 Gα 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 Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, United States.
authors.forename: Ravi
authors.initials: R
authors.lastname: Kant
authors.affiliation: Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, United States.
authors.forename: Baisen
authors.initials: B
authors.lastname: Zeng
authors.affiliation: Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, United States.
authors.forename: Celestine J
authors.initials: CJ
authors.lastname: Thomas
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, United States.
authors.forename: Stephen R
authors.identifier: 0000-0001-6307-6166
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D000818:Animals; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D020662:Guanine Nucleotide Exchange Factors; D018832:Molecular Chaperones; D009687:Nuclear Proteins; C485885:Ric8a protein, rat; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D018631:ras Proteins
keywords: biochemistry; biophysics; heterotrimeric G proteins; hydrogen-deuterium exchange mass spectrometry; non-receptor guanine nucleotide exchange factor; protein dynamics; protein secondary structure; rat; structural biology