The nucleotide exchange factor Ric-8a is a chaperone for the conformationally dynamic nucleotide-free state of gαi1

Celestine J. Thomas, Klára Briknarová, Jonathan K. Hilmer, Navid Movahed, Brian Bothner, John P. Sumida, Gregory G. Tall, Stephen R. Sprang

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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 [ 15N]Gαi1[] bound to Ric-8A shows considerable loss of peak intensity relative to that of [ 15N]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.

Original languageEnglish
Article numbere23197
JournalPLoS ONE
Issue number8
StatePublished - 2011


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