Document Type

Article

Publication Title

Plos One

Publication Date

8-11-2011

Volume

6

Issue

8

Disciplines

Biochemistry | Chemistry | Life Sciences | Physical Sciences and Mathematics

Abstract

Heterotrimeric G protein alpha subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of G alpha, 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 G alpha subunits. Ric-8A binds to G alpha.GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free G alpha i1 is stable, but dissociates upon binding of GTP to G alpha i1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from G alpha i1, experiments were conducted to characterize the physical state of nucleotide-free G alpha i1 (hereafter referred to as G alpha i1[]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free G alpha i1 is more accessible to trypsinolysis than G alpha i1.GDP, but less so than G alpha i1[] alone. The TROSY-HSQC spectrum of [N-15]G alpha i1[] bound to Ric-8A shows considerable loss of peak intensity relative to that of [N-15]G alpha i1.GDP. Hydrogen-deuterium exchange in G alpha i1[] bound to Ric-8A is 1.5-fold more extensive than in G alpha i1.GDP. Differential scanning calorimetry shows that both Ric-8A and G alpha i1.GDP undergo cooperative, irreversible unfolding transitions at 47 degrees and 52 degrees, respectively, while nucleotide-free G alpha i1 shows a broad, weak transition near 35 degrees. The unfolding transition for Ric-8A: G alpha i1[] is complex, with a broad transition that peaks at 50 degrees, suggesting that both Ric-8A and G alpha i1[] are stabilized within the complex, relative to their respective free states. The C-terminus of G alpha i1 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 G alpha.

DOI

10.1371/journal.pone.0023197

Creative Commons License

Creative Commons Attribution 3.0 License
This work is licensed under a Creative Commons Attribution 3.0 License.

Share

COinS