Small Guanine Nucleotide-binding Proteins Research Articles

Crosstalk between Rho of Plants GTPase signalling and plant hormones.

Rho of Plants (ROPs) constitute a plant-specific subset of small guanine nucleotide-binding proteins within the Cdc42/Rho/Rac family. These versatile proteins regulate diverse cellular processes, including cell growth, cell division, cell morphogenesis, organ development, and stress responses. In recent years, the dynamic cellular and subcellular behaviours orchestrated by ROPs have unveiled a notable connection to hormone-mediated organ development and physiological responses, thereby expanding our knowledge of the functions and regulatory mechanisms of this signalling pathway. This review delineates advancements in understanding the interplay between plant hormones and the ROP signalling cascade, focusing primarily on the connections with auxin and abscisic acid pathways, alongside preliminary discoveries in cytokinin, brassinosteroid, and salicylic acid responses. It endeavours to shed light on the intricate, coordinated mechanisms bridging cell- and tissue-level signals that underlie plant cell behaviour, organ development, and physiological processes, and highlights future research prospects and challenges in this rapidly developing field.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS−/−) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS−/− mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS−/− livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition.

Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.

Molecular and functional interactions of alpha-synuclein with Rab3a

Alpha-synuclein (a-Syn) is a presynaptic protein, the misfolding of which is associated with Parkinson’s disease. Rab GTPases are small guanine nucleotide binding proteins that play key roles in vesicle trafficking and have been associated with a-Syn function and dysfunction. a-Syn is enriched on synaptic vesicles, where it has been reported to interact with GTP-bound Rab3a, a master regulator of synaptic vesicle trafficking. a-Syn is known to bind weakly to Rab8a in solution via a positively charged patch, but the physiological implications of such interactions have not been explored. Here, we investigate direct interactions between a-Syn and Rab3a in solution and on lipid membranes using NMR spectroscopy. We find that the C terminus of a-Syn interacts with Rab3a in a manner similar to its previously reported interaction with Rab8a. While weak in solution, we demonstrate that this interaction becomes stronger when the proteins are bound to a membrane surface. The Rab3a binding site for a-Syn is similar to the surface that contacts the Rab3a effector rabphilin-3A, which modulates the enzymatic activity of Rab3a. Accordingly, we show that a-Syn inhibits GTP hydrolysis by Rab3a and that inhibition is more potent on the membrane surface, suggesting that their interaction may be functionally relevant. Finally, we show that phosphorylation of a-Syn residue Ser 129, a modification associated with Parkinson’s disease pathology, enhances its interactions with Rab3a and increases its ability to inhibit Rab3a GTP hydrolysis. These results represent the first observation of a functional role for synuclein-Rab interactions and for a-Syn Ser 129 phosphorylation.

Allostery and dynamics in small G proteins.

The Ras family of small guanine nucleotide-binding proteins behave as molecular switches: they are switched off and inactive when bound to GDP but can be activated by GTP binding in response to signal transduction pathways. Early structural analysis showed that two regions of the protein, which change conformation depending on the nucleotide present, mediate this switch. A large number of X-ray, NMR and simulation studies have shown that this is an over-simplification. The switch regions themselves are highly dynamic and can exist in distinct sub-states in the GTP-bound form that have different affinities for other proteins. Furthermore, regions outside the switches have been found to be sensitive to the nucleotide state of the protein, indicating that allosteric change is more widespread than previously thought. Taken together, the accrued knowledge about small G protein structures, allostery and dynamics will be essential for the design and testing of the next generation of inhibitors, both orthosteric and allosteric, as well as for understanding their mode of action.

Rac1 Nanoscale Organization on the Plasma Membrane Is Driven by Lipid Binding Specificity Encoded in the Membrane Anchor.

Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.

Photo Regulation of Small G-Protein RAS using Photochromic Peptide

The small guanine nucleotide binding proteins (G-protein), which are known as a molecular switch is central regulator of cellular signaling pathway. The regulation mechanism of G-protein is well studied at molecular level. Ras is one of the G-protein which have essential role in cellular signal is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). GTP-Ras induced by GEF is active state, whereas GDP-Ras induced by GAP is inactive state. The conformational change induced by GTP binding enables Ras to transduce a signal into downstream through direct interaction with its effectors. Also, mutant Ras known as constitutive active cause excess signaling transduction to downstream effectors, which is pathogenesis of canceration. In this study, we focused on the interaction between Ras and its GEF, Son of Sevenless (SOS) to control Ras function using photochromic compounds. Previously it was shown that the peptide mimicking the αH-helix, Ras binding region of SOS effectively inhibits Ras function competing with native SOS. Therefore, it is expected that photo-induced structural change of the peptide conjugated with photochromic compound enable us to control Ras activity indirectly. We designed and synthesized several SOS αH-Helix peptides which contains two cysteine residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative, ABDM which change its structure reversibly by UV-VIS irradiation. CD and NMR analysis revealed that the modification of the peptide with ABDM stabilizes secondary structure and UV and VIS light irradiations induce secondary structural change reversibly. SOS dependent nucleotide exchange reaction of Ras in the presence of ABDM-SOS peptide were also altered photo-reversibly. Spontaneous cellular uptake of FITC conjugated peptide was observed by confocal microscopy. We also examined the photoreversible ERK pathway inhibition with ABDM-peptide in HeLa cell.

Towards Piecing Together the RAS-RAF Puzzle: Dynamics of Membrane-Associated KRAS4b-RAF RBD/CRD Ternary Complex

The RAS family of small guanine nucleotide-binding proteins are critical for various cellular signaling pathways and are among the most common culprits in human cancer, with KRAS4b mutations occurring at high rates in pancreatic, lung, and colorectal cancer. Membrane-anchored RAS binds to effectors from the RAF kinase family at two domains: (i) the RAS-binding domain (RBD) whose complex with RAS has been solved experimentally, and (ii) the cysteine-rich domain (CRD) that mutational assays show can bind RAS independently. However, the structure of the RAS/RBD/CRD ternary complex still remains unknown, including its membrane orientation and interactions. We have used a combination of all-atom and coarse-grained molecular dynamics (MD) simulations to investigate these questions, focusing on the complex between KRAS4b and the RBD/CRD domains of BRAF that experiments show is the initially-activated RAF isoform. First, single-domain simulations of CRD or RBD alone indicate their associations with the membrane. Next, binary complex simulations based on the RAS/RBD crystal structure show multiple dominant membrane orientations, where RBD could either be away from the membrane or making direct membrane interactions. Finally, extensive simulations based on computational docking of CRD to the RAS/RBD crystal structure suggest a viable ternary complex model where CRD embeds into the membrane via both of its hydrophobic loops, RAS interacts with the membrane at the surface covering helices 4 and 5 of the G domain, and RBD away from the membrane. We are currently investigating if interactions of KRAS4b with the RBD/CRD domains of the CRAF isoform lead to changes in the ternary complex structure and to differential membrane interactions. These studies should provide insights that will contribute to our understanding of both normal and cancer-activated signaling pathways.

Photo Regulation of Small G-Protein Ras using Photochromic Peptide

The small guanine nucleotide binding protein (G-protein), which are known as a molecular switch is central regulator of cellular signaling pathway. The regulation mechanism of the protein is well studied at molecular level. Ras is one of the G-protein which have essential role in signaling cascade reaction in cell is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). GTP bout Ras induced by GEF is active state and GDP bound Ras induced by GAP is inactive state. The conformational change induced by GTP binding enable Ras to transduce a signal into downstream through direct interaction with its effectors. Also, mutant Ras known as constant active state cause excess signaling transduction to downstream effectors, which is pathogenesis of canceration, In this study, we focused on the interaction between Ras and its GEF, Son of sevenless (SOS) in order to control Ras function using photochromic compounds. Previously it is shown that the peptide mimicking the αH-helix, Ras binding region of SOS effectively inhibits Ras function competing with native SOS. Therefore, it is expected that photo-induced structural change of the peptide conjugate with photo-switch enable us to control Ras activity. We designed and synthesized SOS αH-helix peptide which contains two cysteine residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative, ABDM which change its structure reversibly by UV-VIS irradiation. The peptide modified with ABDM exhibited secondary structural change reversibly upon UV and visible light irradiation. Binding of ABDM-peptide to Ras and SOS dependent nucleotide exchange reaction were also altered photo-reversibly. We also tried to examine the photo-reversible effect of ABDM-peptide for HeLa cell.

Photoregulation of Small G-Proteins using Photochromic Molecules

The small guanine nucleotide binding proteins (G-protein), which are known as a molecular switch, is a central regulator of cellular signal transduction processes leading to transcription, cell cycle progression, growth, migration, cytoskeletal changes, apoptosis, cell survival, and senescence. The switching mechanism has been well studied at the molecular level. It is known that the G-proteins are activated in the GTP bound state and inactivated in the GDP bound state. The conformational change induced by GTP binding enables Ras to transduce a signal downstream through direct interaction with its effectors. The switching of G-protein is controlled by Guanine nucleotide Exchange Factor (GEF) and GTPase Activating Protein (GAP). Firstly, we attempted to control G-proteins RhoA and Ras photo-reversibly by introducing photochromic molecules into the functional regions of G-protein. We prepared RhoA and H-Ras mutants which have a single cysteine residue at the functional site and modified with photochromic azobenzene and spiropyran derivatives. GTPase and nucleotide exchange rate for some of the photochromic molecules modified RhoA and Ras were photo-reversibly altered. We also examined photo-regulated interaction of RhoA modified with photochromic molecule with its target kinase ROCK2. Secondly, we introduce photochromic molecules into the regulatory factor GEF. It is known that αH-helix region of SOS (GEF) is located at the interface to Ras. We designed and synthesized SOS αH-helix peptide which contains two cysteine residues at the cross-linkable positions with bifunctional azobenzene derivative. The peptide was cross-linked with azobenzene di-maleimide intramolecularly. Photo-reversible interaction of the ABDM modified peptide with Ras was examined.

SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling.

Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

Membrane potential regulates growth

Changes in electrical potential across the plasma membrane can affect cell growth. Zhou et al . discovered that membrane potential influenced the organization of phospholipids in the membrane of cultured mammalian cells and neurons in intact flies (see the Perspective by Accardi). This in turn regulated localization and activity of the small guanine nucleotide binding protein K-Ras, an important regulator of cell proliferation. The cell membrane may thus function analogously to a field-effect transistor by adjusting the strength of mitogenic signaling. Y. Zhou, C.-O. Wong, K.-j. Cho, D. van der Hoeven, H. Liang, D. P. Thakur, J. Luo, M. Babic, K. E. Zinsmaier, M. X. Zhu, H. Hu, K. Venkatachalam, J. F. Hancock, Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling. Science 349 , 873–876 (2015). [Abstract][Full Text] A. Accardi, Lipids link ion channels and cancer. Science 349 , 789–790 (2015). [Summary][Full Text]

Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia.

Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

Meta‐Proteomic Analysis of PTMs in G protein Signaling Systems – New Tools and Unsuspected Discoveries

The collective structural and functional data for prototypical G protein signaling (GPS) systems – defined by 7‐transmembrane receptors (GPCRs), heterotrimeric G proteins (Gαβγ) and Regulators of G‐protein Signaling (RGS) – is arguably the most comprehensive of its kind to date. Comparatively little is known about how G protein structure and function is altered by post‐translational modifications (PTMs). Here we report the first structurally‐resolved, meta‐proteomic map of all empirically validated PTMs for GPS proteins. Using a novel PTM analysis tool called Structural Projection of PTMs (SPoP), a total of 1,910 unique eukaryotic PTMs were coalesced onto available structures within the Protein Data Bank. The resulting dataset has enabled the first‐ever visualization of the PTM topology for each member of a prototypical GPS system. By factoring total PTM observations, protein structure, protein interactions and sequence conservation, we estimated a function potential value for each PTM. Rank order comparison of calculated function potentials with literature‐based evidence of function reveals that SPoP has predictive power. Based on these results, we have identified major PTM hotspots in GPS proteins; predicted and tested the involvement of previously unsuspected PTM regulatory elements; and revealed surprising differences between the PTM topologies of large and small guanine‐nucleotide binding proteins. We propose that meta‐proteomic and structural PTM analysis with SPoP can lead to the discovery of functionally important modification sites that regulate biomedically‐relevant classes of signaling molecules.

Overexpression of an adenosine diphosphate-ribosylation factor gene from the halophytic grass Spartina alterniflora confers salinity and drought tolerance in transgenic Arabidopsis

Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms.

Role of Ras isoforms in γδ T-cell development

The small guanine nucleotide binding proteins of the Ras family (H-, K-, and N-ras isoforms) couple surface receptors, including T-cell antigen receptors, to a variety of cellular responses. Ras proteins are highly homologous and expressed ubiquitously, raising questions as to their functional specificity. H-ras and N-ras have been shown to be dispensable for development in the thymus but critical for peripheral Th1 differentiation of αβ T cells [1]. In the γδ T-cell lineage, in contrast, the specific function of Ras isoforms had not been addressed.

ArfGAP1 function in COPI mediated membrane traffic: currently debated models and comparison to other coat-binding ArfGAPs.

The ArfGAPs are a family of proteins containing an ArfGAP catalytic domain that induces the hydrolysis of GTP bound to the small guanine nucleotide binding-protein ADP-ribosylation factor (Arf). Functional models for Arfs, which are regulators of membrane traffic, are based on the idea that guanine nucleotide-binding proteins function as switches: Arf with GTP bound is active and binds to effector proteins; the conversion of GTP to GDP inactivates Arf. The cellular activities of ArfGAPs have been examined primarily as regulatory proteins that inactivate Arf; however, Arf function in membrane traffic does not strictly adhere to the concept of a simple switch, adding complexity to models explaining the role of ArfGAPs. Here, we review the literature addressing the function Arf and ArfGAP1 in COPI mediated transport, focusing on two critical and integrated functions of membrane traffic, cargo sorting and vesicle coat polymerization. We briefly discuss other ArfGAPs that may have similar function in Arf-dependent membrane traffic outside the ER-Golgi.

Oscillatory Dynamics of Cdc42 GTPase in the Control of Polarized Growth

Cells promote polarized growth by activation of Rho-family protein Cdc42 at the cell membrane. We combined experiments and modeling to study bipolar growth initiation in fission yeast. Concentrations of a fluorescent marker for active Cdc42, Cdc42 protein, Cdc42-activator Scd1, and scaffold protein Scd2 exhibited anticorrelated fluctuations and oscillations with a 5-minute average period at polarized cell tips. These dynamics indicate competition for active Cdc42 or its regulators and the presence of positive and delayed negative feedbacks. Cdc42 oscillations and spatial distribution were sensitive to the amounts of Cdc42-activator Gef1 and to the activity of Cdc42-dependent kinase Pak1, a negative regulator. Feedbacks regulating Cdc42 oscillations and spatial self-organization appear to provide a flexible mechanism for fission yeast cells to explore polarization states and to control their morphology.

Membrane Fission: The Biogenesis of Transport Carriers

Membrane-bound transport carriers are used to transfer cargo between membranes of the secretory and the endocytic pathways. The generation of these carriers can be classified into three steps: segregation of cargo away from the residents of a donor compartment (cargo sorting), generation of membrane curvature commensurate with the size of the cargo (membrane budding or tubulation), and finally separation of the nascent carrier from the donor membrane by a scission or membrane fission event. This review summarizes advances in our understanding of some of the best-characterized proteins required for the membrane fission that separates a transport carrier from its progenitor compartment: the large GTPase dynamin, the small guanine nucleotide-binding (G) proteins of the Arf family, BAR (Bin-amphiphysin-Rvs) domain proteins, and protein kinase D. These proteins share their ability to insert into membranes and oligomerize to create the large curvatures; however, the overall process of fission that involves these proteins appears to be quite different.

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.