Nucleotide Binding Site Of Tubulin Research Articles

Promotion of tubulin assembly by poorly soluble taxol analogs

Promotion or inhibition of tubulin assembly into microtubules is the standard in vitro assay for evaluating potential antimicrotubule agents. Many agents to be tested are poorly soluble in aqueous solution and require a cosolvent such as dimethyl sulfoxide (DMSO). However, DMSO itself can promote tubulin assembly, and its inclusion in assays for compounds that induce tubulin assembly complicates interpretation of the results. Substituting GDP for GTP in the exchangeable nucleotide binding site of tubulin produces a less active form of the protein, tubulin–GDP. Here it is shown that tubulin–GDP can be assembled into normal microtubules in DMSO concentrations up to 15% (v/v), and polymerization assays performed under these conditions can be compared with assays run under more standard conditions. Assays for measuring the effective concentration of a ligand for promotion of tubulin assembly (EC 50), measuring the concentration for inhibition of tubulin assembly (IC 50) by a colchicine site ligand, and measuring tubulin critical concentrations in the presence of poorly soluble taxol derivatives are illustrated.

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly.

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin.

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.

An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules.

We have developed a coupled enzyme system composed of hexokinase, glucose, and nucleoside diphosphate kinase which is able to rapidly convert GTP at the exchangeable nucleotide binding site of tubulin to GDP. Using this method, we have studied the dynamic properties of microtubules and tubulin subunits in the presence of GDP. Conversion of GTP to GDP causes microtubules to change to a new, somewhat lower steady state level; dilution studies show that subunits disassemble from microtubules at steady state in the presence of GDP at a rate comparable to that in the presence of GTP; in reactions of existing microtubules with tubulin-GDP subunits it was found that tubulin-GDP subunits do not participate in net microtubule elongation. From these observations we conclude that in the presence of tubulin-GDP the microtubule steady state has an unusual property; in spite of the fact that the microtubule is continuously undergoing rapid subunit loss, but not subunit addition, a nearly constant steady state level of microtubule mass is maintained. This must mean that tubulin-GDP subunits, although unable to participate in a net addition, can participate in additions each of which compensates for the dissociation of a single subunit from the microtubule. This is equivalent to the existence of an isoenergetic exchange of a tubulin-GDP in solution for a subunit which had been lost from the end of a microtubule.

Binding of fluorescent analogs of GTP to the exchangeable nucleotide binding site of tubulin.

6 S tubulin from which the exchangeably bound guanine nucleotide had been removed was examined for its ability to bind two fluorescent analogs of GTP. One analog, (y-AmNS)GTP, contains the fluorophore l-aminonaphthalene 5-sulfonate attached to the y phosphate of GTP via a phosphoamidate bond. The second analog contains the same fluorophore attached to the sulfur atom of GTP-y-S via a 5-atom linker (GTP-y-SF). Both analogs bind to tubulin lacking nucleotide bound to the exchangeable site, as evidenced by significant increases in the anisotropy of nucleotide fluorescence in solutions containing tubulin. The Kd for the tubulinGTP-y-SF complex is 2.3 X M in buffers without glycerol. In solutions containing 25% glycerol both analogs bind about an order of magnitude more tightly (& for GTP-y-SF = 3.2 X M, Kd for (y-AmNS)GTP = 3.9 X M). Analysis of steady state and dynamic fluorescence anisotropies shows that the fluorophore of (y-AmNS)GTP bound to tubulin exhibits little rotational freedom and that the observed depolarization of fluorescence results largely from rotation of the tubulin-nucleotide complex. In contrast, GTP-y-SF bound to tubulin undergoes rapid rotational movement independent of the rotation of the tubulin-nucleotide complex. A number of GTP analogs were examined for their ability to displace tubulin-bound (y-AmNS)GTP, as measured by fluorescence anisotropy. These experiments show that some alterations of the phosphoryl side chain (GTP-y-S and guanosine-5’-(cu,P-methylene) triphosphate) have little effect on binding affinity, whereas others drastically weaken binding (guanosine5’-(P,y-methylene) triphosphate). The &y-methylene analog binds very weakly but is known to be able to promote assembly at concentrations sufficient to saturate the tubulin-exchangeable nucleotide-binding site. In contrast, the GTP analog containing a blocked phosphoryl terminus binds much more avidly than the plymethylene analog but fails to promote assembly. Thus, binding of a guanine nucleoside triphosphate is a necessary, but not sufficient, condition for promotion of tubulin assembly.