title: The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1.
abstract: Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; and.
authors.forename: Ned
authors.initials: N
authors.lastname: Van Eps
authors.affiliation: Center for Biomolecular Structure and Dynamics and Division of Biological Sciences, University of Montana, Missoula, MT 59812.
authors.forename: Celestine J
authors.initials: CJ
authors.lastname: Thomas
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; and hubbellw@jsei.ucla.edu stephen.sprang@umontana.edu.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.affiliation: Center for Biomolecular Structure and Dynamics and Division of Biological Sciences, University of Montana, Missoula, MT 59812 hubbellw@jsei.ucla.edu stephen.sprang@umontana.edu.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D020662:Guanine Nucleotide Exchange Factors; D018631:ras Proteins
keywords: G protein; double electron electron resonance spectroscopy; guanine nucleotide exchange factor; protein dynamics; tertiary structure