Mechanism of GTPase-Activity-Induced Self-Assembly of Human Guanylate Binding Protein 1

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of large GTPases (LGs). In the course of GTP hydrolysis, the protein undergoes structural changes leading to self-assembly of the protein, which is a characteristic property of all family members. For self-assembly, the protein employs two distinct interaction sites, one of which is located within the LG domain of the protein located at the N-terminus, and the second is located in the C-terminal α-helical domain. Here, we identify intramolecular contacts between the LG domain and the helical part of hGBP1, which relay nucleotide-dependent structural changes from the N-terminus to the C-terminus and thereby mediate tetramer formation of the protein through a second contact site at the C-terminus. Furthermore, we demonstrate the impact of this intramolecular communication on the enzymatic activity of hGBP1 and on its cellular localization.     

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition

hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.     

The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis

Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62-Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation.     

Tetramerization of human guanylate-binding protein 1 is mediated by coiled-coil formation of the C-terminal α-helices

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 μM. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.     

Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-gamma-induced hGBP1 (human guanylate binding protein 1)

Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of P(i) formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds.     

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.     

A novel GTP-binding protein hGBP3 interacts with NIK/HGK

A novel human guanylate-binding protein (GBP) hGBP3 was identified and characterized. Similar as the two human guanylate-binding proteins hGBP1 and hGBP2, hGBP3 has the first two motifs of the three classical guanylate-binding motifs, GXXXXGKS (T) and DXXG, but lacks the N (T) KXD motif. Escherichia coli-expressed hGBP3 protein specifically binds to guanosine triphosphate (GTP). Using a yeast two-hybrid system, it was revealed that the N-terminal region of hGBP3 binds to the C-terminal regulatory domain of NIK/HGK, a member of the group I GCK (germinal center kinase) family. This interaction was confirmed by in vitro glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays.     

Characterization of the G alpha(s) regulator cysteine string protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP(1-198) (full-length), CSP(1-112), and CSP(1-82) on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G alpha(s) and increases steady-state GTP hydrolysis. CSP(1-198) modulation of G alpha(s) was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP(1-112) was Hsc70-SGT-independent. CSP(1-112) preferentially associated with the inactive GDP-bound conformation of G alpha(s). Consistent with the stimulation of GTP hydrolysis, CSP(1-112) increased guanine nucleotide exchange of G alpha(s). The interaction of native G alpha(s) and CSP was confirmed by coimmunoprecipitation and showed that G alpha(s) associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the beta2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G alpha(s) and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.     

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.     

p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.     

The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.     

The structure of the TrmE GTP-binding protein and its implications for tRNA modification

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.     

Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase

Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.     

Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.     

Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: implications for GTP hydrolysis

Ras superfamily GTP-binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP-binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP-binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS-GTPases. Analysis of the amino acid sequences of HAS-GTPases reveals prominent presence of insertions around the GTP-binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras-like GTPases. The substituted hydrophobic residue adopts a "retracted conformation," where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS-GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the "retracted conformation" of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain).     

Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

YjeQ,an essential,conserved, uncharacterized protein from Escherichia coli,is an unusual GTPase with circularly permuted G-motifs and marked burst kinetics

The Escherichia coli protein YjeQ represents a protein family whose members are broadly conserved in bacteria and have been shown to be indispensable to the growth of E. coli and Bacillus subtilis [Arigoni, F., et al. (1998) Nat. Biotechnol. 16, 851]. Proteins of the YjeQ family contain all sequence motifs typical of the vast class of P-loop-containing GTPases, but show a circular permutation, with a G4-G1-G3 pattern of motifs as opposed to the regular G1-G3-G4 pattern seen in most GTPases. All YjeQ family proteins display a unique domain architecture, which includes a predicted N-terminal OB-fold RNA-binding domain, the central permuted GTPase module, and a zinc knuckle-like C-terminal cysteine cluster. This domain architecture suggests a possible role for YjeQ as a regulator of translation. YjeQ was overexpressed, purified to homogeneity, and shown to contain 0.6 equiv of GDP. Steady state kinetic analyses indicated slow GTP hydrolysis, with a k(cat) of 9.4 h(-)(1) and a K(m) for GTP of 120 microM (k(cat)/K(m) = 21.7 M(-)(1) s(-)(1)). YjeQ also hydrolyzed other nucleoside triphosphates and deoxynucleotide triphosphates such as ATP, ITP, and CTP with specificity constants (k(cat)/K(m)) ranging from 0.2 to 1.0 M(-)(1) s(-)(1). Pre-steady state kinetic analysis of YjeQ revealed a burst of nucleotide hydrolysis for GTP described by a first-order rate constant of 100 s(-)(1) as compared to a burst rate of 0.2 s(-)(1) for ATP. In addition, a variant in the G1 motif of YjeQ (S221A) was substantially impaired for GTP hydrolysis (0.3 s(-)(1)) with a less significant impact on the steady state rate (1.8 h(-)(1)). In summary, E. coli YjeQ is an unusual, circularly permuted P-loop-containing GTPase, which catalyzes GTP hydrolysis at a rate 45 000 times greater than that of turnover.     

Structural basis for the unique biological function of small GTPase RHEB

The small GTPase Rheb displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human Rheb in complexes with GDP, GTP, and GppNHp (5'-(beta,gamma-imide)triphosphate), which reveal novel structural features of Rheb and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of Rheb undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the alpha-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of Rheb. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in Rheb is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of Rheb as the catalytic residue.     

Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif (¹⁰³DXEKGD¹⁰⁸), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α-³²P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the ¹⁰³DXEKGD¹⁰⁸ motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.