GTP analogues interact with the tubulin exchangeable site during assembly and upon binding

The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that the tightly bound magnesium in tubulin is associated with the N-site GTP

In an attempt to determine whether the tightly bound Mg2+ found in purified tubulin in associated with the N-site GTP or the E-site GDP or GTP, we removed the E-site nucleotide by several means: (i) alkaline phosphatase treatment; (ii) displacement using excess GMPPCP; and (iii) polymerizing tubulin in the presence of alkaline phosphatase and non-hydrolyzable analogues. The Mg2+ content remained equal to about 1 mol/mol tubulin under conditions where denaturation did not occur. Moreover, the Mg/GTP ratio always remained equal to 1. These results indicate that the Mg2+ is associated with the N-site GTP.     

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.     

Assembly of nonneural microtubules in the absence of glycerol and microtubule-associated proteins.

Microtubule protein from Ehrlich ascites tumor cells purified by an in vitro polymerization process in the absence of glycerol and calcium chelators contains several accessory proteins but lacks the high molecular weight proteins which are present in neurotubulin. DEAE-Sephadex chromatography of two-times cycled tubulin removes these nontubulin proteins, resulting in pure tubulin, as critically examined by sodium dodecyl sulfate gel electrophoresis. This tubulin can readily assemble into microtubules in assembly buffer, at low magnesium concentrations, without glycerol and at tubulin concentrations above 0.8 mg/mL. Electron microscopy shows that the tubules are identical with normal microtubules. When the purified tubulin fraction was reduced and carboxymethylated, a significant minor protein component could be observed electrophoretically, migrating between alpha- and beta-tubulin. At present, the identity and function of this protein are not known. The results demonstrate that the in vitro assembly of tubulin from Ehrlich ascites tumor cells does not require high molecular weight proteins or gamma-like factor(s) as has been proposed for the neurotubulin system.     

Irradiation by electrons of tubulin solution in vitro causes tubulin assembly in absence of a magnesium cofactor

It was shown that irradiation of a tubulin solution (5 x 10(-6) mol/l) with electrons makes tubulin assemble to microtubules. The response of the system was monitored by the femtosecond laser technique. The assembly under these conditions occurs without Mg2+ (magnesium cofactor) and GTP. At 730 (the first harmonic) and 365 nm (the second harmonic), a rise in signal intensity occurs during the first 60-70 ns followed by the onset of tubulin assembly to microtubules, which was registered by the methods of spectrophotometry and electron spectroscopy. Theoretically this effect can be explained by the appearance of hydrated electrons in the solvated tubulin shell. Hydrated electrons are mostly in a long-living polaron state, which can be considered as an ensemble of quasi-dipoles of the electron-H3O+ type. The interaction of quasi-dipoles with the weak internal electrostatic field of tubulin leads, due to nonlinear effects, to a manifold rise in the intensity of the electrostatic field of the solvated shell of initially nonpolymerized tubulin chains. Finally, the increased field makes separate tubulin chains aggregate to microtubules. The effect observed is identical to the action of Mg2+ on the GTP site of beta-tubulin, which transfers it to a slightly perturbed triplet state.     

Modulation of the root effect in goldfish by ATP and GTP

Both ATP and GTP are present in considerable amount in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2–6 times more effective than ATP.The two triphosphates account for $\text{3}\text{4}$ of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH.     

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.     

Modulation of the Root effect in goldfish by ATP and GTP.

Both ATP and GTP are present in considerable amounts in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2-6 times more effective than ATP. The two triphosphates account for 3/4 of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH.     

Kinetic analysis of protein kinase C: product and dead-end inhibition studies using ADP, poly(L-lysine), nonhydrolyzable ATP analogues, and diadenosine oligophosphates

The kinetic mechanism of protein kinase C (PKC) was analyzed via inhibition studies using the product MgADP, the nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imidotriphosphate) (MgAMPPNP), the peptide antagonist poly(L-lysine), and several naturally occurring ATP analogues that are produced in rapidly growing cells, i.e., the diadenosine oligophosphates (general structure: ApnA; n = 2-5). By use of histone as the phosphate acceptor, the inhibition of PKC by MgAMPPNP and MgADP was found to be competitive vs MgATP (suggesting that these compounds bind to the same enzyme form), whereas their inhibition vs histone was observed to be noncompetitive. In contrast, the inhibition by poly(L-lysine) appeared competitive vs histone but uncompetitive vs MgATP, which is consistent with a model wherein MgATP binding promotes the binding of poly(L-lysine) or histone. With the diadenosine oligophosphates, the degree of PKC inhibition was found to increase according to the number of intervening phosphates. The diadenosine oligophosphates Ap4A and Ap5A were the most effective antagonists of PKC, with Ap5A being approximately as potent as MgADP and MgAMPPNP. However, as opposed to MgADP and MgAMPPNP, Ap4A and Ap5A appear to act as noncompetitive inhibitors vs both MgATP and histone, suggesting that they can interact at several points in the reaction pathway. These studies support the concept of a steady-state mechanism where MgATP binding preferentially precedes that of histone, followed by the release of phosphorylated substrate and MgADP. Furthermore, these results indicate a differential interaction of the diadenosine oligophosphates with PKC, when compared to other adenosine nucleotides.     

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg²⁺, which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on α-tubulin, based on molecular modeling studies.     

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP reduced inhibition with microtubule-associated proteins and magnesium

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg²⁺, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-β-D-arabinofuranosylguanine-5′-diphosphate (araGDP). Omission of Mg²⁺ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernable relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.     

Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.     

Reexamination of the role of nonhydrolyzable guanosine 5'-triphosphate analogues in tubulin polymerization: reaction conditions are a critical factor for effective interactions at the exchangeable nucleotide site

Recently it was proposed [O'Brien, E. T., & Erickson, H. P. (1989) Biochemistry 28, 1413-1422] that tubulin polymerization supported by guanosine 5'-(beta,gamma-imidotriphosphate) [p(NH)ppG], guanosine 5'-(beta,gamma-methylenetriphosphate) [p(CH2)ppG], and ATP might be due to residual GTP in reaction mixtures and that these nucleotides would probably support only one cycle of assembly. Since we had observed polymerization with these three compounds, we decided to study these reactions in greater detail in two systems. The first contained purified tubulin and a high concentration of glycerol, the second tubulin and microtubule-associated proteins (MAPs). In both systems, reactions supported by nucleotides other than GTP were most vigorous at lower pH values. In the glycerol system, repeated cycles of polymerization were observed with ATP and p(CH2)ppG, but not with p(NH)ppG. With p(NH)ppG, a single cycle of polymerization was observed, and this was caused by contaminating GTP. In the MAPs system, repeated cycles of polymerization were observed with both nonhydrolyzable GTP analogues, even without contaminating GTP, but ATP was not active at all in this system. Binding to tubulin of p(NH)ppG, p(CH2)ppG, and, to a lesser extent, ATP was demonstrated indirectly, since high concentrations of the three nucleotides displaced radiolabeled GDP originally bound in the exchangeable site, with p(NH)ppG the most active of the three compounds in this displacement assay. The failure of GTP-free p(NH)ppG to support tubulin polymerization in our glycerol system even though it displaced GDP from the exchangeable site was further investigated by examining the effects of p(NH)ppG on polymerization and polymer-bound nucleotide with low concentrations of GTP. The two nucleotides appeared to act synergistically in supporting polymerization, so that a reaction occurred with a subthreshold GTP concentration if p(NH)ppG was also in the reaction mixture. Analysis of radiolabeled exchangeable-site nucleotide in polymers formed in reaction mixtures containing both GTP and p(NH)ppG demonstrated that p(NH)ppG which entered polymer did so primarily at the expense of GDP originally bound in the exchangeable site rather than at the expense of GTP. It appears that in the glycerol reaction condition, tubulin-p(NH)ppG cannot initiate tubulin polymerization but that it can participate in polymer elongation. ATP and p(CH2)ppG also entered the exchangeable site during polymerization without GTP in glycerol, as demonstrated by displacement of radiolabeled GDP from polymer when these alternate nucleotides were used.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of the O2(.-)-generating oxidase in plasma membrane from bovine polymorphonuclear neutrophils by arachidonic acid, a cytosolic factor of protein nature, and nonhydrolyzable analogues of GTP

A reconstitution system for activation of the O2(.-)-generating oxidase from bovine polymorphonuclear neutrophils (PMN) is described. This system consisted of three components, namely, a particulate fraction enriched in plasma membrane, a supernatant fluid (cytosolic fraction) recovered by high-speed centrifugation from sonicated resting bovine PMN, and arachidonic acid. The pH optimum (7.8) and the Km value for NADPH (45 microM) of the activated oxidase were virtually the same as those found in the purified enzyme. All three components had to be present during the preincubation for elicitation of oxidase activity. A further enhancement of oxidase activity was observed with the addition of nonhydrolyzable GTP analogues, such as guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imidotriphosphate) (GMP-PNP), to the preincubation medium. In contrast, GDP-beta-S drastically decreased oxidase activation. In a two-stage experiment, a 9-min preincubation of PMN membranes with arachidonic acid and GTP-gamma-S followed by a 1-min contact with the cytosolic fraction led to a more marked activation than did preincubation of the cytosol with arachidonic acid and GTP-gamma-S for 9 min followed by a 1-min contact with membranes, suggesting the presence of a G-protein in the membrane fraction. In the absence of added cations, the reconstitution system exhibited a substantial oxidase activity which was totally prevented by ethylenediaminetetraacetic acid (EDTA). Mg2+ added at a concentration of 0.5-1 mM enhanced oxidase activation by about 30%, indicating that endogenous Mg2+ or other activating cations were sufficient to ensure 70% of maximal activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.     

Turnover of tubulin and the N site GTP in Chinese hamster ovary cells

Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by co-polymerization with nonradioactive porcine brain microtubule protein. 75% of the soluble tubulin in CHO extracts co-polymerizes with the porcine protein through several cycles, without preferential loss of either CHO or porcine subunits. After phosphocellulose chromatography of the co-polymerized microtubules, the CHO tubulin is radiochemically homogeneous, as judged by SDS-polyacrylamide gel electrophoresis. CHO tubulin purified in this way has 1 mole of nucleotide per mole of protein noncovalently bound at the non-exchangeable or N site. This-layer chromatography indicates that the N site nucleotide is entirely ribo-GTP. Label and chase experiments show that the N site GTP exchanges intracellularly with a half-time of 33 hr in growing cells which have a generation time of 17 hr, while the tubulin polypeptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the gamma-phosphate of the N site nucleotide can be detected but occurs very slowly, with a half-time of 24 hr. These results suggest that the N site nucleotide may function in vivo as a stable structural co-factor of the tubulin molecule and render improbable the possibility that it has a regulatory role in microtubule assembly.     

Assembly and turnover of detyrosinated tubulin in vivo

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.     

The effects of magnesium ions on the interactions of ox brain and liver glutamate dehydrogenase with ATP and GTP.

The effects of ATP and GTP on the activities of ox liver and brain glutamate dehydrogenase were determined in the absence and presence of added Mg2+ ions. Although GTP was an inhibitor of the enzyme reaction assayed in the direction of NAD+ reduction, the magnesium complex of this nucleotide had no effect on the activity. Similarly the magnesium complex of ATP was without effect on the activity of the enzyme although the free nucleotide was an activator. These results suggest that it is important to take account of magnesium complex formation when considering the regulatory actions of these nucleotides.