AGS3-C is a noncompetitive inhibitor of Ric-8At-catalyzed exchange of GDP by GTPγS on Gα_i1. Lineweaver-Burk plot of the initial velocities of Ric-8At-catalyzed guanine nucleotide exchange determined as a function of substrate (Gα_i1·GDP) concentration (Inset). Reaction mixtures contained Ric-8At (50 nm), Gα_i1 (0.1–1 μm) and 10 μm GTPγS. Nucleotide binding was monitored by tryptophan fluorescence as indicated in Fig. 3. Experiments were conducted in the absence of AGS3-C (▪), 0.3 μm (▴), or 0.4 μm (•) AGS3-C.

A cycle of nonreceptor-mediated guanine nucleotide binding and hydrolysis by Gα_i1. The cycle includes Gα_i1 (Gα), in the presence of Ric-8A (R), AGS3-C (A), and a hypothetical GAP protein (Gp) (reactions involving the latter are shown in gray, because GAP proteins were not included in the present work). Kinetically reversible reactions are shown with double-headed arrows. The outer arc (labeled 1) describes the reaction, initiated at the point indicated by the perpendicular bar, corresponding to the green FRET traces shown in Fig. 5. The inner circle (labeled 2) describes the cyclic course of the reaction initiated at the point indicated by the perpendicular bar, corresponding to the black FRET trace in Fig. 5, bottom panel.

Isothermal titration calorimetric analysis of the interactions of AGS3-C with Gα_i1·GDP and AGS3-C with Ric-8At:Gα_i1. Fixed aliquots (8 μl) of Gα_i1·GDP (200 μm) were injected into a calorimetric cell containing 1.43 ml of AGS3-C (6 μm), and the heat released was recorded (trace A, thermogram shown in inset). Data were fit by nonlinear least squares to a “two independent sets of sites” model, showing that four molecules of Gα_i1·GDP bind to one molecule of AGS3-C and that binding is cooperative. Shown in trace B, fixed aliquots (8 μl) of AGS3-C (350 μm) were injected into 1.43 ml of Ric-8At:Gαil (10 μm) complex. No significant heats were generated in course of this reaction.

Ric-8At catalyzes GTPγS binding to Gα_i1. Kinetics of Ric-8At-assisted binding of GTPγS to Gα_i1 was followed by tryptophan fluorescence. 400 μl of Gα_i1·GDP (1 μm) was equilibrated for 10–15 min at 25 °C in a fluorescence cuvette. 10-Fold excess of GTPγS was added to Gα_i1, and fluorescence at 340 nm upon excitation at 290 nm was monitored in the absence or presence of Ric-8At (1, 0.75, 0.5, 0.25, 0.125, and 0 μm; black traces from top to bottom). A similar experiment under the same conditions and in the presence of 1 μm Ric-8A was conducted with the W211A mutant of Gα_i1 (blue trace). Inset, tryptophan fluorescence at the 2-min time points of the reaction as a function of increasing Ric-8At concentration.

FRET detection of transient ternary complex formation by YFP-Ric-8A with CFP-AGS3-C:Gα_i1·GDP. Top panel, evolution and decay of FRET emission resulting from the interaction of YFP-Ric-8A with CFP-AGS3-C: Gα_i1·GDP. 120 μl of YFP-Ric-8A (55 μm) was mixed rapidly with an equal volume of CFP-AGS3:Gα_i1·GDP (12 μm) in a 20-μl cell of a pneumatically driven stopped-flow apparatus. FRET emission fluorescence at 527 nm upon excitation at 415 nm was measured for 300 s (green trace). A control experiment was performed by rapidly mixing equal volumes (120 μl) of YFP-Ric-8A (55 μm) with CFP-AGS3-C (12 μm) under the same reaction conditions (black trace). Traces shown are averages of 12–15 independent experiments performed under identical conditions. GDP release from AGS3-C:Gα_i1·GDP (50 μl, 12 μm) was monitored by tryptophan fluorescence emission (340 nm) after rapid mixing with 50 μl of Ric-8At (55 μm, red trace, or 250 μm, blue trace). Bottom panel, evolution of FRET because of addition of GTP to a reaction mixture identical to that shown in the top panel at the 150-s time point. Equal volumes of YFP-Ric-8A (110 μl, 55 μm) and CFP-AGS3-C:Gα_i1·GDP (12 μm complex) were rapidly mixed into an aging loop. After 150 s, GTP (110 μl, 150 μm) was then mixed with 110 μl of the aging loop contents into the optical cell of the stopped-flow apparatus (note that, because of the 1:4 stoichiometry of the CFP-AGS3-C:Gα_i1·GDP complex, the concentration of GTP injected into the optical cell is in ∼3-fold excess to Gα_i1). FRET emission at 527 nm was measured for 1300 s. The green trace represents the reaction sequence depicted in top panel, and the black trace shows the evolution of FRET emission because of association of CFP-AGS3-C:Gα_i1·GDP with YFP-Ric-8A upon GTP binding and hydrolysis by Gα_i1. Arrow indicates the time point at which GTP was added to the aged reaction mixture. The FRET emission base line for first reaction sequence at 150 s (green trace) was aligned with that for the second phase of the reaction (black trace).

Ric-8At induces dissociation of AGS3-C:Gα_i1·GDP, whereas AGSC-3 does not disrupt the Ric-8At:Gα_i1 complex. A, schematic representation of the protein constructs used in this study. Vertical hatched boxes represent tetratricopeptide repeats; gray boxes, GPR/GoLoco repeats in AGS3. Residue numbers at N- and C-terminal boundaries of constructs are indicated. B, gel-filtered AGS3-C:His₆Gα_i1·GDP complex (25 μm) was incubated for 2 h at 20 °C with Ric-8At (100 μm) and then bound to 50 μl of Ni²⁺ IMAC resin. The resin was run on an SDS-polyacrylamide gel, and the proteins were visualized by staining with Coomassie Brilliant Blue dye (lane 1). Gel-filtered Ric-8At:His₆Gα_i1 complex (25 μm) was incubated for 2 h at 20 °C with AGS3-C (250 μm) and analyzed as above (lane 2). Similar results were obtained in the presence or absence of excess GDP. Lanes 3–7 indicate the proteins AGS3 (lane 3), His₆-Gα_i1 (lane 4), and Ric-8 (lane 5) and protein complexes AGS3-C:His₆-Gα_i1·GDP (lane 6) and Ric-8At:His₆-Gα_i1 (lane 7) used in this study. C, AGS3-C:Gα_i1·GDP complex (14 μm) was incubated for 2 h at 20 °C with Ric-8At (100 μm) in a total volume of 1 ml, and the reaction mixture was resolved using a tandem Superdex 200/75 gel filtration matrix (profile I). Conversely, Ric-8At:Gα_i1 (100 μm) complex was incubated with AGS3-C (200 μm) for 2 h at 20 °C and treated under similar gel filtration conditions (profile II). Inset, the peak fractions for both experiments were analyzed by SDS-PAGE to visualize the proteins. Insets show Coomassie stained SDS-polyacrylamide gels corresponding to peaks from profiles I and II. Peaks and corresponding lanes for profile I are labeled 1–5 and for profile II labeled A–D.