title: The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.
abstract: Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.
authors.affiliation: Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana, United States of America.
authors.forename: Celestine J
authors.initials: CJ
authors.lastname: Thomas
authors.forename: Klára
authors.initials: K
authors.lastname: Briknarová
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Hilmer
authors.forename: Navid
authors.initials: N
authors.lastname: Movahed
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.forename: John P
authors.initials: JP
authors.lastname: Sumida
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Tall
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D000818:Animals; D041961:Deuterium Exchange Measurement; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011522:Protons; D051381:Rats; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
chemical_list: C522851:Gnai1 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D009711:Nucleotides; D011522:Protons; C485885:Ric8a protein, rat; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014357:Trypsin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go