Inactive GDP-bound Conformation Research Articles

Adagrasib: A landmark in the KRASG12C-mutated NSCLC.

Adagrasib: A landmark in the KRASG12C-mutated NSCLC.

Pharmacophoric analogs of sotorasib-entrapped KRAS G12C in its inactive GDP-bound conformation: covalent docking and molecular dynamics investigations.

For decades, KRAS G12C was considered an undruggable target. However, in recent times, a covalent inhibitor known as sotorasib was discovered and approved for the treatment of patients with KRAS G12C-driven cancers. Ever since the discovery of this drug, several preclinical efforts have focused on identifying novel therapeutic candidates that could act as covalent binders of KRAS G12C. Despite these intensive efforts, only a few KRAS G12C inhibitors have entered clinical trials. Hence, this highlights the need to develop effective drug candidates that could be used in the treatment of KRAS G12C-driven cancers. Herein, we embarked on a virtual screening campaign that involves the identification of pharmacophores of sotorasib that could act as covalent arsenals against the KRAS G12C target. To our knowledge, this is the first computational study that involves the compilation of sotorasib pharmacophores from an online chemical database against KRAS G12C. After this library of chemical entities was compiled, we conducted a covalent docking-based virtual screening that revealed three promising drug candidates (CID_146235944, CID_160070181, and CID_140956845) binding covalently to the crucial nucleophilic side chain of Cys12 and interact with the residues that form the cryptic allosteric pocket of KRAS G12C in its inactive GDP-bound conformation. Subsequently, ADMET profiling portrayed the covalent inhibitors as lead-like candidates, while 100ns molecular dynamics was used to substantiate their stability. Although our overall computational study has shown the promising potential of the lead-like candidates in impeding oncogenic RAS signaling, more experimental efforts are needed to validate and establish their preclinical relevance. Implication of KRAS G12C in cancer and computational approach towards impeding the KRAS G12C RAS signaling.

Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

Computer-aided drug design of some KRAS G12C inhibitors: Targeting the covalent and allosteric binding site for cancer therapy

KRAS is one of the most investigated anticancer targets today, owing to its common mutations in a variety of lethal malignancies. KRAS G12C has been the focus of cancer research since the identification of the druggable allosteric binding site located in close vicinity with G12C mutation. Herein, we present a computer-assisted drug discovery strategy for discovering new and selective KRAS G12C covalent inhibitors. With the screening of an online database that holds millions of assayed and curated compounds targeted against KRAS G12C, a total of 1001 compounds were generated. Furthermore, these ligands were subjected to a ligand-filtering process to identify those with an acrylamide warhead group that could bind covalently with the Cys12 nucleophile of KRAS G12C and like sotorasib, the only clinically approved drug against the therapeutic cancer target till date. The resulting 202 compounds were then subjected to a covalent-docking virtual screening experiment, leading to the identification of three putative binders (CID_13728717, CID_146235508, and CID_146235420) against the KRAS G12C in its inactive GDP-bound conformation. Subsequently, ADMET modeling and molecular dynamics investigations showed why the compounds may be regarded as drug-like and stable covalent binders of KRAS G12C, respectively. In particular, the molecular enumeration profile of CID_13728717 was the most satisfactory, excelling more than the standard sotorasib in this computationally driven drug discovery research. Hence, it is most likely to emerge as an effective KRAS G12C drug after extensive experimental efforts.

Abstract 1058: Inhibition of nuclear transport protein XPO1 potentiates the effect of KRASG12C inhibitors

Abstract Introduction: The discovery of compounds that can bind covalently to KRASG12C and lock it in its inactive GDP-bound conformation has opened a window of opportunity to selectively target KRASG12C. Such agents have shown promising results in preclinical tumor models and recently in clinical trials as well. However, factors such as feedback reactivation or bypass of KRAS dependence are known to limit antitumor activity of KRAS inhibitors. This necessitates the need for combination approaches that can potentially sensitize tumors to KRAS inhibitors when co-targeted. In this study, we have tested two potent KRASG12C inhibitors, MRTX1257 and AMG510, on PDAC and NSCLC cell lines both as single agents and also in combination with nuclear transport inhibitor, Selinexor (KPT-330/XPOVIO). Methods: Cell growth inhibition was determined by MTT assay. Drug synergy analysis was performed and the isobolograms were generated using CalcuSyn 2.1 software (Biosoft, Cambridge, UK). Spheroid disintegration assay, clonogenic assay, immunofluorescence assay, and Western blotting were performed by standard methods. Results: MRTX1257 and AMG510, when combined with KPT-330, synergistically inhibited (Combination Index values less than 1) the proliferation of KRASG12C mutant cancer cell lines, namely MiaPaCa-2 (PDAC) and NCI-H2122 (NSCLC). Such an effect was observed neither in KRASWT (NCI-H1650) nor KRASG12D mutant (Panc-1) cells, suggesting that the synergistic effect on cell viability was specific to KRASG12C. Combined treatment of KPT-330 with either MRTX1257 or AMG510 resulted in enhanced disintegration of MiaPaCa-2 and NCI-H2122 spheroids, indicating the efficacy of the drug combinations in a 3-D cell growth model. In addition, similar synergistic action was also seen in a clonogenic assay, where MRTX1257 and AMG510 reduced the number and size of colonies formed by MiaPaCa-2 cells and KPT-330 further augmented this effect. KPT-330 also potentiated the MRTX1257- or AMG510-induced inhibition of KRAS-dependent signaling targets such as ERK1/2 phosphorylation. Mechanistically, we observed that KPT-330 caused nuclear retention of ETS repressor factor (ERF), a transcriptional repressor that is exported upon phosphorylation by ERK. Given that XPO1 transports hundreds of proteins, we are currently evaluating the global changes in RAS pathway proteins using high throughput approaches. Such studies are anticipated to unveil the molecular underpinnings of the effectiveness of this co-targeting approach that would help advance this combination of nuclear transport inhibitor with KRASG12C inhibitors to be tested in the clinic. Conclusion: In conclusion, we have demonstrated that the inhibitor of nuclear transport protein XPO1 has the ability to potentiate the anticancer activity of KRASG12C inhibitors in in vitro preclinical models of PDAC and NSCLC. Citation Format: Husain Y. Khan, Md. Hafiz Uddin, Yue Zhang, Yosef Landesman, Ammar Sukari, Misako Nagasaka, Asfar S. Azmi. Inhibition of nuclear transport protein XPO1 potentiates the effect of KRASG12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1058.

A phase I/II multiple expansion cohort trial of MRTX849 in patients with advanced solid tumors with KRAS G12C mutation.

TPS3161 Background: RAS proteins are part of the family of small GTPases which regulate intracellular signaling pathways responsible for cell growth, migration, survival and differentiation. Oncogenic point mutations in RAS in codons 12, 13, and 61 occur in up to one-third of all human cancers and result in constitutive activation of RAS signaling, playing a key role in uncontrolled cellular growth and malignant transformation. Mutant KRASG12C in particular comprises approximately 14% of lung adenocarcinoma and 4% of colon adenocarcinoma, and less commonly in certain other types of cancer. For decades, KRAS was considered undruggable due to its high affinity for GTP/GDP and the lack of a clear binding pocket. Recent discoveries have enabled the development of compounds, including MRTX849, that covalently bind to KRASG12C at the cysteine at residue 12, lock the protein in its inactive GDP-bound conformation, and inhibit KRAS-dependent signal transduction. MRTX849 is a potent, orally-available, mutation-selective small molecule covalent inhibitor of KRASG12C. MRTX849 inhibits KRASG12C signaling in cell lines harboring this mutation, and results in tumor regression in a broad spectrum of KRASG12C animal models. Methods: This multi-center, Phase 1/2, multiple expansion cohort trial evaluates the safety, pharmacokinetics (PK), metabolites, pharmacodynamics, and clinical activity of MRTX849 in patients with advanced solid tumor malignancies with a KRAS (p.G12C) mutation. The study starts with an evaluation of dose and regimen of MRTX849 using a combination of the accelerated titration and modified toxicity probability interval designs, with MRTX849 initially administered once daily in a continuous regimen expressed in 3-week cycles. As potentially viable regimens are identified, Phase 1b expansion cohorts will be opened to provide greater safety and PK data for determination of the recommended Phase 2 dose (RP2D) and regimen. In Phase 2, separate cohorts of patients by histological diagnosis, including non-small cell lung cancer, colorectal, and other solid tumors, will be enrolled and evaluated for clinical activity using a predictive probability design. The study is open for enrollment, and recruitment is ongoing. Clinical trial information: NCT03785249.

PARP-1-dependent RND1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin

RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, RND1, is rapidly induced by camptothecin. RND1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases RND1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces RND1 transcription. In addition, overexpression of RND1 increases PARP-1, suggesting a cross-talk between PARP-1 and RND1. Finally, RND1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight RND1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-RND1 pathway protects cells against apoptosis induced by camptothecin.

Regulation of RabGAPs involved in insulin action.

Rab (Ras-related proteins in brain) GTPases are key proteins responsible for a multiplicity of cellular trafficking processes. Belonging to the family of monomeric GTPases, they are regulated by cycling between their active GTP-bound and inactive GDP-bound conformations. Despite possessing a slow intrinsic GTP hydrolysis activity, Rab proteins rely on RabGAPs (Rab GTPase-activating proteins) that catalyze GTP hydrolysis and consequently inactivate the respective Rab GTPases. Two related RabGAPs, TBC1D1 and TBC1D4 (=AS160) have been described to be associated with obesity-related traits and type 2 diabetes in both mice and humans. Inactivating mutations of TBC1D1 and TBC1D4 lead to substantial changes in trafficking and subcellular distribution of the insulin-responsive glucose transporter GLUT4, and to subsequent alterations in energy substrate metabolism. The activity of the RabGAPs is controlled through complex phosphorylation events mediated by protein kinases including AKT and AMPK, and by putative regulatory interaction partners. However, the dynamics and downstream events following phosphorylation are not well understood. This review focuses on the specific role and regulation of TBC1D1 and TBC1D4 in insulin action.

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner.

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.

Computational analysis for GNAQ mutations: New insights on the molecular etiology of Sturge-Weber syndrome

Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gβ subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.

High-Throughput Dual Screening Method for Ras Activities and Inhibitors.

Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.

Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase)

RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

Novel topography of the Rab11-effector interaction network within a ciliary membrane targeting complex

Small GTPases function as universal molecular switches due to the nucleotide dependent conformational changes of their switch regions that allow interacting proteins to discriminate between the active GTP-bound and the inactive GDP-bound states. Guanine nucleotide exchange factors (GEFs) recognize the inactive GDP-bound conformation whereas GTPase activating proteins (GAPs), and the GTPase effectors recognize the active GTP-bound state. Small GTPases are linked to each other through regulatory and effector proteins into functional networks that regulate intracellular membrane traffic through diverse mechanisms that include GEF and GAP cascades, GEF-effector interactions, common effectors and positive feedback loops linking interacting proteins. As more structural and functional information is becoming available, new types of interactions between regulatory proteins, and new mechanisms by which GTPases are networked to control membrane traffic are being revealed. This review will focus on the structure and function of the novel Rab11-FIP3-Rabin8 dual effector complex and its implications for the targeting of sensory receptors to primary cilia, dysfunction of which causes cilia defects underlying human diseases and disorders know as ciliopathies.

The RhoGAP ARHGAP19 controls cytokinesis and chromosome segregation in T lymphocytes

Small GTP-binding proteins of the Rho family orchestrate the cytoskeleton remodelling events required for cell division. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) promote cycling of Rho GTPases between the active GTP-bound and the inactive GDP-bound conformations. We report that ARHGAP19, a previously uncharacterised protein, is predominantly expressed in hematopoietic cells and has an essential role in the division of T lymphocytes. Overexpression of ARHGAP19 in lymphocytes delays cell elongation and cytokinesis. Conversely, silencing of ARHGAP19 or expression of a GAP-deficient mutant induces precocious mitotic cell elongation and cleavage furrow ingression, as well as excessive blebbing. In relation to these phenotypes, we show that ARHGAP19 acts as a GAP for RhoA, and controls recruitment of citron and myosin II to the plasma membrane of mitotic lymphocytes as well as Rock2-mediated phosphorylation of vimentin, which is crucial to maintain the stiffness and shape of lymphocytes. In addition to its effects on cell shape, silencing of ARHGAP19 in lymphocytes also impairs chromosome segregation.

Pan‐Selective Aptamers for the Family of Small GTPases

Pan‐Selective Aptamers for the Family of Small GTPases

Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

Evolutionary expansion of the Ras switch regulatory module in eukaryotes

Ras proteins control many aspects of eukaryotic cell homeostasis by switching between active (GTP-bound) and inactive (GDP-bound) conformations, a reaction catalyzed by GTPase exchange factors (GEF) and GTPase activating proteins (GAP) regulators, respectively. Here, we show that the complexity, measured as number of genes, of the canonical Ras switch genetic system (including Ras, RasGEF, RasGAP and RapGAP families) from 24 eukaryotic organisms is correlated with their genome size and is inversely correlated to their evolutionary distances from humans. Moreover, different gene subfamilies within the Ras switch have contributed unevenly to the module’s expansion and speciation processes during eukaryote evolution. The Ras system remarkably reduced its genetic expansion after the split of the Euteleostomi clade and presently looks practically crystallized in mammals. Supporting evidence points to gene duplication as the predominant mechanism generating functional diversity in the Ras system, stressing the leading role of gene duplication in the Ras family expansion. Domain fusion and alternative splicing are significant sources of functional diversity in the GAP and GEF families but their contribution is limited in the Ras family. An evolutionary model of the Ras system expansion is proposed suggesting an inherent ‘decision making’ topology with the GEF input signal integrated by a homologous molecular mechanism and bifurcation in GAP signaling propagation.

Identification and Characterization of a Peptidic Ligand for Ras

The development of new ligands for the oncoprotein Ras can provide tools for the study of this important signaling component or potentially serve as therapeutic agents for the treatment of Ras-associated diseases. Herein, we report a peptidic Ras ligand identified through naïve phage display. Panning a phage library with a diversity of 10(9) transormants successfully identified a peptide dodecamer that contains two internal consensus motifs and binds Ras in both the active GTP- and inactive GDP-bound conformations with low micromolar dissociation constants. The dodecamer does not alter the intrinsic GTPase activity of Ras, does not compete for Ras binding to the Ras binding domain of Raf, and does not alter cell viability. This novel Ras ligand has the potential to serve in the development of higher-affinity ligands and chemical tools targeting Ras.

Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Biophysical Analysis of the Interaction of Rab6a GTPase with Its Effector Domains

Rab GTPases are key regulators of intracellular vesicular transport that control vesicle budding, cargo sorting, transport, tethering, and fusion. In the inactive (GDP-bound) conformation, Rab GTPases are targeted to intracellular compartments where they are converted into the active GTP-bound form and recruit effector domain containing proteins. Rab6a has been implicated in dynein-mediated vesicle movement at the Golgi apparatus and shown to interact with a plethora of effector proteins. In this study, we identify minimal Rab6a binding domains of three Rab6a effector proteins: PIST, BicaudalD2, and p150(glued). All three domains are >15-kDa fragments predicted to form coiled-coil structures that display no sequence homology to each other. Complex formation with BicaudalD2 and p150 has a moderate inhibitory effect on the intrinsic GTPase activity of Rab6a, while interaction with PIST has no influence on the hydrolysis rate. The effectors bind activated Rab6a with comparable affinities with K(d) values ranging from high nanomolar to low micromolar. Transient kinetic analysis revealed that effectors bind to Rab6a in an apparent single-step reaction characterized by relatively rapid on- and off-rates. We propose that the high off-rates of Rab6.effector complexes enable GTPase-activating protein-mediated net dissociation, which would not be possible if the off-rate were significantly slower.