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 [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.
Highlights
As members of the Ras superfamily of regulatory GTP binding proteins, heterotrimeric G protein a subunits (Ga) undergo cycles of activation and deactivation driven by binding and hydrolysis of GTP [1]
G protein-coupled receptors (GPCRs) act on plasma membrane-localized G protein heterotrimers that consist of GDP-bound Ga tightly associated with heterodimers of Gb and Gc subunits
Circular dichroism (CD) spectroscopy, heteronuclear NMR spectroscopy, hydrogen-deuterium exchange mass spectrometry (HD-MS), and differential scanning calorimetry (DSC), we have found that Ric-8A stabilizes Gai1 in a conformationally dynamic and heterogeneous state which, we propose, facilitates GDP release and subsequent GTP binding
Summary
As members of the Ras superfamily of regulatory GTP binding proteins, heterotrimeric G protein a subunits (Ga) undergo cycles of activation and deactivation driven by binding and hydrolysis of GTP [1]. The structural events that accompany GEFcatalyzed nucleotide exchange on small, Ras-like G proteins are relatively well understood [2], the mechanism of heterotrimeric G protein activation remains enigmatic. Agonist-activated, transmembrane G protein-coupled receptors (GPCRs) [3] are the best characterized heterotrimeric G protein GEFs. GPCRs act on plasma membrane-localized G protein heterotrimers that consist of GDP-bound Ga tightly associated with heterodimers of Gb and Gc subunits. Members of a family of predominantly cytosolic proteins, typified by mammalian Ric-8A, were identified as non-receptor GEFs that catalyze nucleotide exchange directly on Ga subunits of the Gi/o/q/12/13 families [4]. Ric-8A paralogs act in GPCR-independent pathways to orient mitotic spindles in asymmetric cell division, as demonstrated in
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