The N-terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor

The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal guanine nucleotide exchange factor (GEF) domain. In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.     

The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30 degrees C and displayed equilibrium binding constants of 1.2 and 0.5 microM, respectively, in the presence of Mg(2+). In the absence of Mg(2+), the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively. N-Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg(2+) was extremely rapid (k(d) = 1.4 and 1.5 s(-1), respectively), 10(3)- to 10(5)-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg(2+). Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 x 10(-4) s(-1) corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.     

The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase

CgtA(E)/Obg(E)/YhbZ is an Escherichia coli guanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtA(E) with guanine nucleotides and show that its properties are similar to those of the Caulobacter crescentus homolog CgtA(C). CgtA(E) binds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtA(E) is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtA(E) copurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtA(E) and SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtA(E) is involved in the SpoT-mediated stress response.     

Biochemical properties of the Vibrio harveyi CgtAV GTPase

Bacteria encode a number of relatively poorly characterized GTPases, including the essential, ribosome-associated Obg/CgtA proteins. In contrast to Ras-like proteins, it appears that the Obg/CgtA proteins bind guanine nucleotides with modest affinity and hydrolyze GTP relatively slowly. We show here that the Vibrio harveyi CgtA(V) exchanges guanine nucleotides rapidly and has a modest affinity for nucleotides, suggesting that these features are a universal property of the Obg/CgtA family. Interestingly, CgtA(V) possesses a significantly more rapid GTP hydrolysis rate than is typical of other family members, perhaps reflecting the diversity and specificity of bacterial ecological niches.     

Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins.

We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans. This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role. In this report, we describe the isolation and sequence of the cgtA gene. The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family. Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth. Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C. crescentus cell cycle.     

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.     

Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis.

An essential guanine nucleotide-binding protein, Obg, of Bacillus subtilis has been characterized with respect to its enzymatic activity for GTP. The protein was seen to hydrolyze GTP with a Km of 5.4 microM and a kcat of 0.0061 min-1 at 37 degrees C. GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 microM at 37 degrees C. The dissociation constant for GDP from the Obg protein was 0.5 microM at 4 degrees C and was estimated to be 1.3 microM at 37 degrees C. Approximately 80% of the purified protein was capable of binding GDP. In addition to hydrolysis of GTP, Obg was seen to autophosphorylate with this substrate. Subsequent release of the covalent phosphate proceeds at too slow a rate to account for the overall rate of GTP hydrolysis, indicating that in vitro hydrolysis does not proceed via the observed phosphoamidate intermediate. It was speculated that the phosphorylated form of the enzyme may represent either a switched-on or a switched-off configuration, either of which may be normally induced by an effector molecule. This enzyme from a temperature-sensitive mutant of Obg did not show significantly altered GTPase activity at the nonpermissive temperature.     

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as several medical conditions. Individual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94 × 10⁻³ min⁻¹ for DRG1 and 1.36 × 10⁻³ min⁻¹ for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.     

The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA(C) protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA(C) does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA(C) to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA(C). Assays of epitope-tagged wild-type and mutant variants of CgtA(C) indicate that the C terminus of CgtA(C) is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA(C) alleles to function in vivo. Depletion of CgtA(C) led to perturbations in the polysome profile, raising the possibility that CgtA(C) is involved in ribosome assembly or stability.     

Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

The effects on tubulin polymerization and associated guanosine triphosphate hydrolysis of aluminum ion, fluoride and fluoroaluminate species

The effects of aluminum ion, fluoride, and fluoroaluminate species on the assembly of tubulin in the presence of guanine nucleotides and the consequences of these ions on the associated GTPase of microtubules was investigated. Combinations of GDP and fluoroaluminate species were incapable of activating tubulin for polymerization, in contrast to other guanine nucleotide binding proteins, in which these species produce a functional GTP equivalent. Fluoride alone has an effect on GTP-magnesium-promoted microtubule assembly, causing an increased amount of polymer formation and a reduced rate of associated GTP hydrolysis. It is concluded that aluminum ion and fluoroaluminate species possess distinct mechanisms in inhibiting GTP hydrolysis of GTP-binding proteins and that subpopulations of GTP-binding proteins must exist based on differential sensitivities to these ions.     

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.     

An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism

Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange.     

Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Obg subfamily of bacterial GTP-binding proteins: essential proteins of largely unknown functions that are evolutionarily conserved from bacteria to humans

Members of the Obg subfamily of small GTP-binding proteins (called Obg, CgtA, ObgE or YhbZ in different bacterial species) have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Although serious changes in phenotypes are observed in mutant bacteria devoid of Obg or its homologues, specific roles of these GTP-binding proteins remain largely unknown. Recent genetic and biochemical studies, as well as determination of the structures of Obg proteins from Bacillus subtilis and Thermus thermophilus, shed new light on the possible functions of the members of the Obg subfamily and may constitute a starting point for the elucidation of their exact biological role.     

An open conformation of switch I revealed by Sar1-GDP crystal structure at low Mg2+

Mg2+ is essential for guanosine triphosphatase activity and plays key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. To understand the structural basis for Mg2+ function during the GDP/GTP exchange process, we determined the crystal structure of Delta9-Sar1-GDP at low Mg2+ concentration at 1.8A. Two Sar1-GDP molecules in the crystal form a dimer with Mg2+ presenting only in molecule B but not in molecule A. The absence of Mg2+ induces significant conformational changes in the switch I region in molecule A that shows similarities with those of Ha-Ras bound to Sos. The current structure reveals an important regulatory role for Mg2+. We suggest that guanine nucleotide exchange factor may utilize this feature to generate an open conformation for GDP/GTP exchange. Furthermore, we propose a mechanism for COPII assembly and disassembly in which dimerization of Sar1 plays an important role.     

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.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

GTP gamma S inhibits organelle transport along axonal microtubules

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide- specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S- sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.     

GTP-mediated macromolecular interactions: the common features of different systems

G proteins that serve to transduce external signals in membranes share with protein synthesis factors and tubulin structural and functional features that are common to proteins that participate in reversible GTP-mediated macromolecular interactions. These proteins can bind GTP and GDP with high affinity, adopting different structures depending on whether they are complexed with the nucleotide diphosphate or triphosphate. The GTP.protein complex has high affinity for an acceptor macromolecule (or complex of macromolecules) and interacts with it, affecting its activity. These GTP-binding proteins also possess an intrinsic GTPase activity that is generally stimulated by its interaction with the acceptor. The GTPase activity converts the bound GTP to GDP, switching the configuration of the complexed protein to one of low affinity for the acceptor and causing its dissociation. The protein.GDP complex must exchange its GDP for GTP to allow the protein to acquire the high-affinity structure that can cycle back to the acceptor macromolecule. This exchange of guanine nucleotides requires in several instances exchange factors that can regulate the whole process. A detailed comparison of the features of the different systems is made with respect to structural similarities, regulation by protein phosphorylation, ADP ribosylation by bacterial toxins, and requirements for exchange factors. It is also proposed that there is a similar mechanism that involves ATP/ADP-binding proteins.