期刊
PLOS ONE
卷 6, 期 8, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0023197
关键词
-
资金
- National Institutes of Health [DK46371, RR16455, RR020185, RR24237, GM088242]
- The Murdock Charitable Trust
- Washington State Life Science [2496490]
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.
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