The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.

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

Nucleotide-free Gαi1 is relatively unstructured in comparison to Gαi1•GDP, but regains helical secondary structure in the complex with Ric-8A.

<p>Circular dichroic spectra were normalized as mean residue elipticity, and predicted secondary structure assignments are: Ric-8A, <i>red</i>: 87% α-helix; Ric-8A:Gαi1[ ], <i>black</i>: 87% α-helix, 0.5% β-strand; Gαi1•GDP, <i>green</i>: 51% α-helix, and 11% β-strand; Gαi1[ ], <i>blue</i>, 38% α-helix, 9% β-strand.</p

GEF activity of purified Ric-8A fragments defined by limited trypsinolysis and secondary structure analysis.

<p>(A) Coomassie-stained SDS PAGE analysis of Ric-8A after trypsinization for the times indicated below each lane; unique fragments are identified by colored asterisks. (B) Electrospray mass spectrometric analysis of Ric-8A tryptic digest fragments extracted from the SDS PAGE gel shown in panel A; peaks identified by asterisks refer to corresponding bands shown in panel A. Fragment masses (Da) are indicated at each peak position. (C) Amino acid sequence of rat Ric-8A; cylinders indicate helical segments predicted using JPRED <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Cole1" target="_blank">[51]</a>. Residue codes colored red indicate sites of proteolytic cleavage (see panel A). Residue codes in green indicate N or C-termini of recombinant Ric-8A fragments engineered to coincide approximately with proteolytic sites or predicted secondary structure boundaries: ΔC492 denotes the Ric-8A fragment comprising residues 1–492. Both N-terminal truncations ΔN12 and ΔN38 were also C-terminally truncated at residue 492 and comprised residues 12–492 and 38–492, respectively. (D) Kinetics of intrinsic (open symbols) or Ric-8A-stimulated (filled symbols) GDP release (squares) from, or GTPγ binding to (circles) myristoylated Gαi1 were determined by a filter binding assay using radiolabeled nucleotides as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Tall1" target="_blank">[4]</a>. Upper left panel, Gαi1 (200 nM) nucleotide binding and release in the presence of full-length Ric-8A (200 nM); lower left panel, ΔC492Ric-8A (200 nM); upper right panel, ΔC453Ric-8A (200 nM); lower right panel, Gαi1 alone. Data for each panel are normalized to maximum GDP released or GTPγS bound in a single experiment. Data points represent the average of three experiments; standard deviation from the mean is <10%. Time course of GTPγS binding in the absence of Ric-8A, shown at lower right, is replicated in the other panels for comparison. (E), Histogram showing relative rates of Gαi1 GDP release (red bars) and GTPγS binding (blue bars) catalyzed by Ric-8A and Ric-8A truncation mutants (200 nM). Error bars represent +/− one standard deviation of the apparent first-order rate constants determined in three replicates.</p

Intrinsic and Ric-8A-catalyzed GTPγS binding rates of the Gαi1 proteins used in this study.

<p>Intrinsic and Ric-8A-catalyzed kinetics of binding of GTPγS to wild-type Gαi1, W258A-Gαi1, NΔ25Gαi1, GαiCΔ9 and Gαi1-GαsC12 were measured using a fluorescence binding assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Thomas1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Higashijima1" target="_blank">[47]</a>. 400 µl of protein (1 µM) in the GDP bound form was equilibrated for 10–15 min at 25°C in a cuvette. A 10-fold excess of GTPγS was added and fluorescence at 340 nm upon excitation at 290 nm was monitored in the absence (open bars) or presence (filled bars) of Ric-8A (1 µM). Error bars represent +/− one standard deviation apparent first-order rate constants determined in three replicates.</p

¹H-¹⁵N TROSY-HSQC spectrum of Gαi1 shows extensive peak broadening and intensity loss upon binding of Ric-8A.

<p>(A) ¹H-¹⁵N HSQC-TROSY spectra were acquired for [¹⁵N]Gαi1•GDP, and (B) Ric8A:[¹⁵N]Gαi1[ ]. (C) After acquisition of the Ric-8A:[¹⁵N]Gαi1[ ] spectrum, a five molar excess of GTPγS was added to induce dissociation of Ric-8A and formation of Gαi1•GTPγS. After a short incubation, the free Ric-8A was removed by adsorption to IMAC resin, and the ¹H-¹⁵N TROSY-HSQC spectrum of the sample was recorded. Protein concentration, acquisition and processing parameters and contour levels in all panels are the same.</p

Nucleotide-free Gαi1 bound to Ric-8A exhibits rapid hydrogen/deuterium exchange kinetics relative to the Gαi1•GDP complex.

<p>(A) Mass distribution for Gαi1•GDP measured at fixed time points (see panel C) after dilution into D₂O; the mass distribution at the zero time point, before exchange was initiated, corresponds to the red peak centered at 40.1 kDa. The average of the Gαi1 mass distribution increases as H/D exchange reaction proceeds. (B) Mass distribution for Gαi1[ ] in complex with Ric-8A measured at fixed time points after dilution into D₂O; note that the Gαi1[ ] mass distribution becomes multimodal as the H/D exchange reaction proceeds. (C) The increase in mass (Da), determined at the centroid of the mass distribution of Gαi1•GDP (black squares) and Gαi1[ ] derived from the complex with Ric-8A (red circles) is plotted as a function of time after rapid dilution from aqueous buffer into D₂O.</p

Ric-8A provides limited protection of nucleotide-free Gαi1 from trypsin digestion.

<p>Samples were incubated with TPCK treated trypsin at a 1∶1000 molar ratio (trypsin∶sample) at 4°C, withdrawn at the indicated time points, separated by SDS-PAGE and visualized by Coomassie blue staining. (A) Gαi1•GDP: lanes from left to right: molecular weight markers, M; untreated Gαi1•GDP, U; samples digested for 10 and 25 minutes. (B) nucleotide-free Gαi1[ ]: markers, M; untreated Gαi1[ ], U; samples digested for 5 and 10 minutes. Mass spectroscopic analysis identifies band 1 as Gαi1 residues 21–179: observed/calculated mass 17,761/17,774 Da. (C) Ric-8A: markers, M; untreated, U. (D) Ric-8A: after 5, 10 and 15 minutes of trypsin digestion. Mass analysis identifies band 1 as Ric-8A residues 1–408: 46,218/46,207 Da; band 2, residues 72–378: 34,815/34,799; band 3, residues 141–348: 23,834/23,804 Da; band 4, residues 62–178: 13,563/13,523 Da. (E) Ric-8A: Gαi1[ ] complex: markers, M; untreated Ric-8A:Gαi1[ ] complex; R and G indicate bands for intact Ric-8A and Gαi1, respectively; Ric-8A:Gαi1[ ] complex digested for 10 and 25 minutes. Mass analysis identifies band 1 as Ric-8A residues 141–348: 23,834/23,804 Da (present also as band 3 in Panel D); band 2, Gαi1 residues 17–191: 19,646/19,652 Da; band 3, Gαi1 residues 21–179: 17,753/17,761 Da (present as band 1 in panel B); band 4: Gαi1 residues 10–141: 14,532/14,520 Da.</p