Structure of the p115RhoGEF rgRGS domain-Galpha13/i1 chimera complex suggests convergent evolution of a GTPase activator

p115RhoGEF, a guanine nucleotide exchange factor (GEF) for Rho GTPase, is also a GTPase-activating protein (GAP) for G12 and G13 heterotrimeric Galpha subunits. The GAP function of p115RhoGEF resides within the N-terminal region of p115RhoGEF (the rgRGS domain), which includes a module that is structurally similar to RGS (regulators of G-protein signaling) domains. We present here the crystal structure of the rgRGS domain of p115RhoGEF in complex with a chimera of Galpha13 and Galphai1. Two distinct surfaces of rgRGS interact with Galpha. The N-terminal betaN-alphaN hairpin of rgRGS, rather than its RGS module, forms intimate contacts with the catalytic site of Galpha. The interface between the RGS module of rgRGS and Galpha is similar to that of a Galpha-effector complex, suggesting a role for the rgRGS domain in the stimulation of the GEF activity of p115RhoGEF by Galpha13.     

Distinct regions of Galpha13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs

Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

Mapping the Galpha13 binding interface of the rgRGS domain of p115RhoGEF

Structural requirements for function of the Rho GEF (guanine nucleotide exchange factor) regulator of G protein signaling (rgRGS) domains of p115RhoGEF and homologous exchange factors differ from those of the classical RGS domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS domain was undertaken to determine its functional interface with the Galpha(13) subunit. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Galpha(13) and the interactions of RGS4 and RGS9 with their Galpha substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Lack of sequence conservation within the N terminus may also explain the lack of GTPase-activating protein (GAP) activity in a subset of the rgRGS domains. For all mutations, loss of functional GAP activity is paralleled by decreases in binding to Galpha(13). The same mutations, when placed in the context of the p115RhoGEF molecule, produce deficiencies in GAP activity as observed with the rgRGS domain alone but show no attenuation of the regulation of Rho exchange activity by Galpha(13). This suggests that the rgRGS domain may serve a structural or allosteric role in the regulation of the nucleotide exchange activity of p115RhoGEF on Rho by Galpha(13).     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Structure of the rgRGS domain of p115RhoGEF

p115RhoGEF, a guanine nucleotide exchange factor for Rho GTPase, is also a GTPase activating protein (GAP) for G(12) and G(13) heterotrimeric G alpha subunits. Near its N-terminus, p115RhoGEF contains a domain (rgRGS) with remote sequence identity to RGS (regulators of G protein signaling) domains. The rgRGS domain is necessary but not sufficient for the GAP activity of p115RhoGEF. The 1.9 A resolution crystal structure of the rgRGS domain shows structural similarity to RGS domains but possesses a C-terminal extension that folds into a layer of helices that pack against the hydrophobic core of the domain. Mutagenesis experiments show that rgRGS may form interactions with G alpha(13) that are analogous to those in complexes of RGS proteins with their G alpha substrates.     

Identification of critical residues in G(alpha)13 for stimulation of p115RhoGEF activity and the structure of the G(alpha)13-p115RhoGEF regulator of G protein signaling homology (RH) domain complex

RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).     

Protein kinase Calpha-induced p115RhoGEF phosphorylation signals endothelial cytoskeletal rearrangement

Heterotrimeric G-proteins of the Galpha12/13 family activate Rho GTPase through the guanine nucleotide exchange factor p115RhoGEF. Because Rho activation is also dependent on protein kinase Calpha (PKCalpha), we addressed the possibility that PKCalpha can also induce Rho activation secondary to the phosphorylation of p115RhoGEF. Studies were made using human umbilical vein endothelial cells in which we addressed the mechanisms of PKCalpha-induced Rho activation and its consequences on actin cytoskeletal changes. We observed that PKCalpha associated with p115RhoGEF within 1 min of thrombin stimulation and p115RhoGEF phosphorylation was dependent on PKCalpha. Inhibition of PKCalpha-dependent p115RhoGEF phosphorylation prevented the thrombin-induced Rho activation, indicating that the response occurred downstream of PKCalpha phosphorylation of p115RhoGEF. The regulator of G-protein signaling domain of p115RhoGEF, a GTPase activating protein for G12/13, also prevented thrombin-induced Rho activation, indicating the parallel requirement of G12/13 in signaling Rho activation via p115RhoGEF. These data demonstrate a pathway of Rho activation involving PKCalpha-dependent phosphorylation of p115RhoGEF. Thus, Rho activation in endothelial cells and the subsequent actin cytoskeletal re-arrangement require the cooperative interaction of both G12/13 and PKCalpha pathways that converge at p115RhoGEF.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Copper and zinc inhibit Galphas function: a nucleotide-free state of Galphas induced by Cu2+ and Zn2+

The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu(2+) and Zn(2+) inhibit the steady-state GTPase activity of the alpha subunit of GTP-binding protein (Galpha(s)) but do not alter its intrinsic GTPase activity. Cu(2+) and Zn(2+) decrease steady-state GTPase activity by inhibiting the binding of GTP to Galpha(s). Moreover, Cu(2+) and Zn(2+) increase GDP dissociation from Galpha(s) and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) that is already bound to the Galpha(s). Because of their ability to inhibit GTPgammaS binding, preincubation of Cu(2+) or Zn(2+) with Galpha(s) does not permit GTPgammaS to activate Galpha(s) and stimulate AC activity. However, preincubation of Galpha(s) with GTPgammaS followed by addition of Cu(2+) or Zn(2+) did not alter the ability of Galpha(s) to stimulate AC activity. Interestingly, AlF(4)(-) partially restored the ability of Galpha(s), which had been preincubated with Cu(2+) or Zn(2+), to stimulate AC; AlF(4)(-) does not permit the re-association of unbound GDP with Galpha(s). Thus, the interaction of AlF(4)(-) with the nucleotide-free Galpha(s) is sufficient to activate AC. Using antibodies to the N and C termini of Galpha(s), we show that the Cu(2+) interaction site on the G protein is in the C terminus. We conclude that Cu(2+) and Zn(2+) generate a nucleotide-free state of Galpha(s) and that, in the absence of any nucleotide, the gamma-phosphate mimic of GTP, AlF(4)(-), alters Galpha(s) structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu(2+) and Zn(2+) as compared with forskolin-stimulated activity is consistent with Galpha(s) being a primary target of these cations in regulating the signaling from receptor to AC.     

Motion of carboxyl terminus of Galpha is restricted upon G protein activation. A solution NMR study using semisynthetic Galpha subunits

The carboxyl terminus of the G protein alpha subunit plays a key role in interactions with G protein-coupled receptors. Previous studies that have incorporated covalently attached probes have demonstrated that the carboxyl terminus undergoes conformational changes upon G protein activation. To examine the conformational changes that occur at the carboxyl terminus of Galpha subunits upon G protein activation in a more native system, we generated a semisynthetic Galpha subunit, site-specifically labeled in its carboxyl terminus with 13C amino acids. Using expressed protein ligation, 9-mer peptides were ligated to recombinant Galpha(i1) subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Galpha(i1) subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Galpha(i1). To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to Galpha subunits containing 13C labels at the corresponding sites in Galpha(i1): Leu-348 (uniform), Gly-352 (alpha carbon), and Phe-354 (ring). In the GDP-bound state, the spectra of the ligated carboxyl terminus appeared similar to the spectra obtained for 13C-labeled free peptide. Upon titration with increasing concentrations of AlF4-, the 13C resonances demonstrated a marked loss of signal intensity in the semisynthetic Galpha subunit but not in free peptide subjected to the same conditions. Because AlF4- complexes with GDP to stabilize an activated state of the Galpha subunit, these results suggest that the Galpha carboxyl terminus is highly mobile in its GDP-bound state but adopts an ordered conformation upon activation by AlF4-.     

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.     

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.     

Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation

G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP-bound form. Together these results suggest conformational changes occur in the amino-termini of Galpha(i) proteins upon subunit dissociation and upon activating conformational changes. These solution studies reveal insights into conformational changes that occur dynamically in solution.     

Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).     

Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.