Identification of nucleotide-dependent Gα_i-binding peptides via phage display. (A) Representative phage ELISA results indicating the identification of (i) GDP-selective, (ii) GTPγS-selective, and (iii) nucleotide state-independent Gα_i1-binding peptides. (B) Sequences of 12 isolated peptides with GDP-selective binding to Gα_i1, sharing a consensus TWX^E/_DFL motif with the particular peptide used in this study: KB-752. (C) Nucleotide-dependent binding of KB-752 as measured by surface plasmon resonance (SPR). 5 μM Gα_i1 protein (“analyte”), in each of three nucleotide bound states, was injected over immobilized, biotinylated KB-752. Non-specific binding to a control peptide was subtracted from each curve. (D) GDP-bound Gα_i1 was injected at each indicated concentration over immobilized KB-752 to determine the dissociation constant (K_d) for this interaction pair. SPR-derived dissociation constants for the interaction of KB-752 with Gα_i1 and Gα_o, in their ground state (GDP-bound), transition state-mimetic (GDP·AlF₄^- bound), and activated state (GTPγS-bound) forms, were obtained from analyses (n = 4-6 for each state) similar to that shown in panel D. Dissociation constants of >1000 μM were obtained for both Gα subunits in their GDP·AlF₄^--bound form, and for Gα_o bound to GTPγS.

KB-752 is a selective guanine nucleotide exchange factor for Gα_i subunits. (A) KB-752 (10 μM) enhances the GTPγS binding rate of Gα_i1·GDP (50 nM); rate constants at 30 °C: Gα_i1 alone = 0.029 ± 0.006 min^-1, Gα_i1 + KB-752 = 0.086 ± 0.008 min^-1. (B) KB-752 is equipotent as a GEF on all three Gα_i members. 50 nM Gα_i1, Gα_i2, or Gα_i3 was incubated with indicated concentrations of KB-752 and the amount of [³⁵S]GTPγS binding was measured after 10 min at 30°C, expressed as percent of maximal GTPγS binding. KB-752 does not alter the rate of GTPγS binding by (C) Gα_i-heterotrimer Gα_i2·GDP/Gβ₁γ₂ (peptide and protein amounts as in panel A), nor (D) isolated Gα_o·GDP. For the dose-response curve of panel (D), 50 nM Gα_i1 or Gα_o was incubated in the presence of the indicated concentrations of KB-752 and the amount of [³⁵S]GTPγS binding was measured (after 10 min at 30 °C for Gα_i1; after 5 min at 20 °C for Gα_o) as described in Experimental Procedures and is expressed as the percent of GTPγS bound in the absence of KB-752. The EC₅₀ value for GEF activity on Gα_i1 was 5.6 ± 1.1 μM. Data shown are from a representative experiment of 3-5 independent experiments.

KB-752 GEF activity increases steady-state GTP hydrolysis by Gα_i1, but does not act on a Gα_i1 point mutant (R144A) with accelerated spontaneous nucleotide release. (A) 200 nM of wild-type (wt) Gα_i1, R144A Gα_i1, or wild-type Gα_o was added to cuvettes containing 1 μM BODIPY-FL-GTPγS. Real-time nucleotide binding (Kimple et al., 2004) was measured at 25 °C as an increase in fluorescence response (λ_ex = 485 nm; λ_em = 530 nm; slit widths of 3.0 nm) upon binding BODIPY-FL-GTPγS. Mutation of arginine 144 to alanine (R144A) resulted in Gα_i1 nucleotide binding kinetics indistinguishable from that of Gα_o, as previously reported by Remmers and colleagues (Remmers et al., 1999). (B) KB-752 does not alter the rate of GTPγS binding by the mutant Gα_i1 subunit (R144A) with accelerated spontaneous nucleotide exchange comparable to that of wildtype Gα_o (experiment performed as in Figure 2A except conducted at 20 °C). (C) Confirming the GEF activity of KB-752 on wild type Gα_i1, addition of KB-752 (100 μM) to Gα (200 nM) enhances the steady-state hydrolysis of [γ³²-P]GTP by Gα_i1, but has no effect on Gα_o. Note that the rate-limiting step in steady-state hydrolysis of GTP by Gα subunits is release of product (i.e., GDP) and not the hydrolysis of GTP per se (Ross, 2002).

Stereoview of experimental electron density for the KB-752 peptide bound to Gα_i1. The region highlighted is the entire peptide density (model in red; labels in white) found between switch II (α2 helix) and the α3 helix of Gα_i1 (model in green; labels in yellow). Shown is a 2F_o-F_c simulated annealing composite omit map (generated with 5% overall model omitted) contoured at 1 s with electron density shown in white cage.

Biochemical confirmation of the overall structural features of the Gα_i1/KB-752 interaction. (A) Ribbon trace of KB-752 (red) bound between the α2 (“switch II”) and α3 helices of the Gα_i1 Ras-like domain (blue). No contacts are made between KB-752 and the all-helical domain (yellow) or bound GDP (magenta). Switch regions are denoted in green. (B) Structural basis for nucleotide selective binding of KB-752 to Gα_i1. KB-752 peptide (red, translucent) binds Gα_i1 between switch II and the α3 helix; the conformations of these two helices are shown for Gα_i1·GDP/KB-752 (green), Gα_i1·GTPγS (yellow), and Gα_i1·GDP·AlF₄^- (magenta). Whereas the α3 helix is not significantly altered, switch II is displaced to accommodate KB-752 binding. Switch II in both Gα_i1·GTPγS and Gα_i1·GDP·AlF₄^- assumes an extended α-helical conformation that is stabilized relative to Gα_i1·GDP (Mixon et al., 1995; Sprang, 1997). This conformation of switch II is not permissive to KB-752 binding as it creates extensive steric hindrance. In particular, W211 of switch II (shown in space filling) is in a restrictive position relative to W5 of KB-752. (C) The GoLoco motif of RGS14 (orange) is also seen to bind, in an alpha-helical conformation, between switch II and the α3 helix of Gα_i1 (PDB ID 1KJY); highlighted within the C α-carbon ribbon trace of the GoLoco peptide. Other features are colored as in panel A. (D) KB-752 GEF activity does not rely on the all-helical domain. 100 nM of Gα_i1 or a chimeric α containing the Ras-like domain of Gα_i1 and the all-helical domain of Gα_o (“Gαioi”; ref. (Remmers et al., 1999) was incubated in the absence or presence of 50 μM KB-752 and [³⁵S]GTPγS binding after 10 min at 30 °C was measured as described in Experimental Procedures. Data are expressed as percent GTPγS bound relative to Gα protein in the absence of KB-752 (“Control”) and are the average ± SEM of 4 independent experiments. (E) The KB-752 binding site on Gα_i1 overlaps that of GoLoco motif peptides. Gα_i1 (50 nM) was incubated in the absence or presence of the indicated concentrations of a peptide representing the GoLoco motif of RGS12 (R12GL) (Kimple et al., 2002). GTPγS binding was then measured in the presence of the indicated concentrations of KB-752. Data are expressed as fmol of GTPγS bound above that measured in the absence of KB-752 and are from a representative experiment of 3 independent experiments. (F) The binding of KB-752 has no effect on the kinetics of Gα_i1 activation by AlF₄^-, unlike the slowed activation rate seen upon GoLoco peptide binding. Gα_i1-CFP (200 nM) and YFP-RGS4 (280 nM) fusion proteins, previously shown to generate increased fluorescence resonance energy transfer (FRET) upon Gα_i1 activation by AlF₄^- and subsequent RGS-box binding (Willard et al., 2004), were mixed together and pre-incubated with either 10 μM KB-752 peptide or 5 μM GoLoco consensus peptide (AGS3Con; Kimple et al., 2002), prior to the addition of NaF and AlCl 3 to final concentrations of 20 mM and 30 μM, respectively, at the 150-second mark.

Biochemical confirmation of specific interactions between KB-752 and Gα_i1. (A) Positions of KB-752 residues W5, F8, and E11 relative to residues in the switch II and α3 helices of Gα_i1. W5 and F8 are placed within hydrophobic pockets formed by Gα_i1 residues F215, L249, and I253, and W211, I212, and F215, respectively. E11 forms a salt bridge with R208 of Gα_i1. (B) Peptide residues T4 and D7 of the conserved TWX^E/_DFL binding motif form an intrapeptide hydrogen bond network that helps to orient W5 and F8. (C, D) Effects of W5A, F8A, and E11L mutations to KB-752 activity. (C) Indicated KB-752 mutant or wildtype (wt) peptides were each immobilized to a density of ∼1000 RUs on separate streptavidin-coated flow cells and 50 μM GDP-bound Gα_i1 (“analyte”) was injected simultaneously over all four surfaces. (D) Compared to the increase in GTPγS binding observed by addition of 50 μM wildtype KB-752 to 100 nM Gα_i1·GDP, substantial reduction of GEF activity is seen upon mutation to the W5, F8, or E11 residue of KB-752.

Conformational changes in Gα switch regions induced by KB-752 binding. (A,B) Relative orientations of the three switch regions (SI — III) in heterotrimeric (PDB code 1GP2; blue in panel A), Gα_i1·GDP/R14GL (PDB code 1KJY; orange in panel B), and Gα_i1·GDP/KB-752 (green). Movement of switch II (α2 helix) and the connected β3/α2 loop away from the nucleotide binding pocket in Gα_i1·GDP/KB-752 is thought to contribute to GEF activity by creating a route for GDP release. The RGS14 GoLoco motif peptide (R14GL) displaces switch II but does not significantly alter the position of the β3/α2 loop. (C) Binding of KB-752 displaces switch II resulting in a reorientation of the β3/α2 loop away from the guanine nucleotide pocket. KB-752 (red) stabilizes the β3/α2 loop (green) via several hydrogen bonds (indicated as yellow dashes), including the carbonyl oxygen of G202 (Gα) with the indole nitrogen of W5 (KB-752), and the side chain hydroxyl and main chain amide nitrogen of S206 (Gα) with the main chain amide nitrogen and carbonyl oxygen of V3 (KB-752), respectively. A water molecule (magenta ball) is coordinated by both Gα and KB-752 contacts.

Comparison of switch regions and core catalytic residues of KB-752-bound Gα_i1 with other states of Gα_i1. (A) Movement of switch I in the Gα_i1·GDP/KB-752 complex (green), versus its position in the Gα_i1β₁γ₂ heterotrimer (blue) and the Gα_i1·GDP/R14GL complex (orange), results in disruption of a salt-bridge (black dotted line) between R178 and E43 that normally stabilizes bound GDP (magenta) within Gα_i1 when complexed to a GDI (Gβγ or GoLoco peptide). (B) Electron density of the R178 side-chain in the Gα_i1·GDP/KB-752 complex (from a 2Fo-Fc simulated annealing composite omit map contoured to a level of 1 σ) is denoted by white mesh. In the background is the beta-phosphate of the bound GDP (βP). (C,D) Switch region comparisons with activated Gα_i1 states. Switch regions of Gα_i1·GDP/KB-752 (green), Gα_i1·GDP·AlF₄^- (PDB code 1GFI; magenta; panel C), and Gα_i1·GTPγS (PDB code 1GIA; yellow; panel D), are shown along with the residues critical for GTP hydrolysis (R178 and T181 within switch I and Q204 within switch II). GDP from the Gα_i1·GDP/KB-752 structure is shown for reference in each case. Overall conformation of the switch regions of Gα_i1·GDP·AlF₄^- and Gα_i1·GTPγS are very similar, save for key changes in the position of catalytic residue side chains (Wall et al., 1998). Whereas switch I of Gα_i1·GDP/KB-752 is very similar to that of the activated forms, both switch II and III are dramatically removed from the guanine nucleotide to allow for GDP release. The catalytic Q204 residue within switch II is far removed from the bound nucleotide and active site for GTP hydrolysis in the Gα_i1·GDP/KB-752 structure. However, R178 and T181 of switch I are in a strikingly similar position to that of the Gα_i1·GDP·AlF₄^- structure.