Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy

The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The same number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 0.49 and rate constants k(1--1) which are much smaller than 40 s(-1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be observed with equilibrium constants K(1)(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected.     

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.     

Sequential action of two GTPases to promote vacuole docking and fusion

Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions. Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking. Here we explore the roles of the GDP and GTP states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase-activating protein for Ypt7p:GTP), GTPgammaS or GppNHp (non-hydrolyzable nucleotides), and mutant forms of Ypt7p that favor either GTP or GDP states. GDP-bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only the GTP-bound state allows docking. Ypt7p is converted to the GTP-bound state after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p-mediated release of Ypt7p. Ypt7p extraction does not inhibit the Ca(2+)-triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTPgammaS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, two GTPases sequentially govern vacuole docking and fusion.     

Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins

The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.     

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.     

Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.     

Relation between the conformational heterogeneity and reaction cycle of Ras: molecular simulation of Ras

Ras functions as a molecular switch by cycling between the active GTP-bound state and the inactive GDP-bound state. It is known experimentally that there is another GTP-bound state called state 1. We investigate the conformational changes and fluctuations arising from the difference in the coordinations between the switch regions and ligands in the GTP- and GDP-bound states using a total of 830 ns of molecular-dynamics simulations. Our results suggest that the large fluctuations among multiple conformations of switch I in state 1 owing to the absence of coordination between Thr-35 and Mg²⁺ inhibit the binding of Ras to effectors. Furthermore, we elucidate the conformational heterogeneity in Ras by using principal component analysis, and propose a two-step reaction path from the GDP-bound state to the active GTP-bound state via state 1. This study suggests that state 1 plays an important role in signal transduction as an intermediate state of the nucleotide exchange process, although state 1 itself is an inactive state for signal transduction.     

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.     

Simulation of the S2 state multiline electron paramagnetic resonance signal of photosystem II: a multifrequency approach.

The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.     

Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy

The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras. In the present study we investigated structural changes of H-Ras in its di- and triphosphate form that occur upon glucosylation of the effector domain amino acid threonine-35 by LT. (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides. In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center. In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinct conformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination. This conformation is known to have a low affinity to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.     

Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.     

Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.     

Electron-nuclear double resonance of copper complexes of human transferrin

Electron-nuclear double resonance (ENDOR) spectroscopy has been used to study ligand and copper hyperfine interactions in Cu(II) complexes of human transferrin. A nearly isotropic superhyperfine interaction of the Cu(II) spin with a single 14N nucleus was identified, and the principal values of its tensor were estimated. All principal values of the copper hyperfine tensor were also directly measured for the first time. Resonances from at least two exchangeable protons were observed, but their origin could not be ascertained. At physiological pH, and in the presence of bicarbonate, ENDOR spectra of the two metal-binding sites were virtually indistinguishable.     

High resolution crystal structures of human Rab5a and five mutants with substitutions in the catalytically important phosphate-binding loop

GTPase domain crystal structures of Rab5a wild type and five variants with mutations in the phosphate-binding loop are reported here at resolutions up to 1.5 A. Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP+AlF(3), and authentic GTP, respectively. The other variant crystals were obtained in complexes with a non-hydrolyzable GTP analog, GppNHp. All structures were solved in the same crystal form, providing an unusual opportunity to compare structures of small GTPases with different catalytic rates. The A30P mutant exhibits dramatically reduced GTPase activity and forms a GTP-bound complex stable enough for crystallographic analysis. Importantly, the A30P structure with bound GDP plus AlF(3) has been solved in the absence of a GTPase-activating protein, and it may resemble that of a transition state intermediate. Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the catalytic Gln(79) residue and independent of A30P mutation-induced local alterations in the P-loop. The structures suggest an important catalytic role for a P-loop backbone amide group, which is eliminated in the A30P mutant, and support the notion that the transition state of GTPase-mediated GTP hydrolysis is of considerable dissociative character.     

Mechanism of the guanine nucleotide exchange reaction of Ras GTPase--evidence for a GTP/GDP displacement model

Ras GTPases function as binary switches in the signaling pathways controlling cell growth and differentiation by cycling between the inactive GDP-bound and the active GTP-bound states. They are activated through interaction with guanine nucleotide exchange factors (GEFs) that catalyze the exchange of bound GDP with cytosolic GTP. In a conventional scheme, the biochemical roles of GEFs are postulated as stimulating the release of the bound GDP and stabilizing a nucleotide-free transition state of Ras. Herein we have examined in detail the catalyzed GDP/GTP exchange reaction mechanism by a Ras specific GEF, GRF1. In the absence of free nucleotide, GRF1 could not efficiently stimulate GDP dissociation from Ras. The release of the Ras-bound GDP was dependent upon the concentration and the structure of the incoming nucleotide, in particular, the hydrophobicity of the beta and gamma phosphate groups, suggesting that the GTP binding step is a prerequisite for GDP dissociation, is the rate-limiting step in the GEF reaction, or both. Using a pair of fluorescent guanine nucleotides (N-methylanthraniloyl GDP and 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-GTP) as donor and acceptor probes, we were able to detect fluorescence resonance energy transfer between the incoming GTP and the departing GDP on Ras under controlled kinetic conditions, providing evidence that there may exist a novel intermediate of the GEF-Ras complex that transiently binds to two nucleotides simultaneously. Furthermore, we found that Ras was capable of binding pyrophosphate (PPi) with a dissociation constant of 26 microM and that PPi and GMP, but neither alone, synergistically potentiated the GRF1-stimulated GDP dissociation from Ras. These results strongly support a GEF reaction mechanism by which nucleotide exchange occurs on Ras through a direct GTP/GDP displacement model.     

Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal

The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.     

Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.Ras-like small G-proteins are ubiquitously expressed, conserved molecular switches that couple extracellular signals to various cellular responses. Different signals can activate GEFs² that induce the small G-protein to switch from the inactive, GDP-bound state to the active, GTP-bound state. This induces a conformational change that allows downstream effector proteins to bind specifically to and be activated by the GTP-bound protein to mediate diverse biological responses. Small G-proteins are returned to the GDP-bound state by hydrolyzing GTP with the help of GAPs. Ras (Ha-Ras, Ki-Ras, and N-Ras) and Rap proteins (Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C) have similar effector-binding regions that interact predominantly with RA domains or the structurally similar RBDs present in a variety of different proteins. Both protein families operate in different signaling networks. For instance, Ras is central in a network controlling cell proliferation and cell survival, whereas Rap1 predominantly controls cell adhesion, cell junction formation, cell secretion, and cell polarity. These different functions are reflected in a largely different set of GEFs and GAPs. Also the downstream effector proteins operate in a selective manner in either one of the networks.     

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.     

Conformational states of ADP ribosylation factor 1 complexed with different guanosine triphosphates as studied by 31P NMR spectroscopy

Guanine nucleotide binding proteins (GNB-proteins) play an essential role in cellular signaling, acting as molecular switches, cycling between the inactive, GDP-bound form and the active, GTP-bound form. It has been shown that conformational equilibria also exist within the active form of GNB-proteins between conformational states with different functional properties. Here we present (31)P NMR data on ADP ribosylation factor 1 (Arf1), a GNB-protein involved in Golgi traffic, promoting the coating of secretory vesicles. To investigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety of commonly used GTP analogues, namely, GppCH(2)p, GppNHp, and GTPγS, were analyzed. To gain deeper insight into the conformational state of active Arf1, we titrated with Cu(2+)-cyclen and GdmCl and formed the complex with the Sec7 domain of nucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, from (31)P NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This state seems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effector domain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward a conformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchange factors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effector interacting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuning mechanism of regulation and thereby an interesting target for the modulation of protein activity.     

Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification

A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch.