The Active GTP- and Ground GDP-Liganded States of Tubulin Are Distinguished by the Binding of Chiral Isomers of Ethyl 5-Amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl Carbamate

Abstract

NSC 613862 (S)-(-) and NSC 613863 (R)-(+) are the two chiral isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3, 4-b]pyrazin-7-yl carbamate. Both compounds bind to tubulin in a region that overlaps the colchicine site. They induce formation of abnormal polymers from purified GTP-Mg-tubulin, the active assembly form of tubulin, in glycerol-free buffer with magnesium [De Ines, C., Leynadier, D., Barasoain, I., Peyrot, V., Garcia, P., Briand, C., Rener, G. A., and Temple, C., Jr. (1994) Cancer Res. 54, 75-84]. In this study, we observed that the S-isomer can promote polymerization of GDP-tubulin, the inactive assembly-incompetent form of tubulin, into nonmicrotubular structures at a critical protein concentration of 1 mg/mL (12 mM MgCl2). Neither the R-isomer nor colchicine have this ability. By electron microscopy, these tubulin polymers showed the same poorly defined filamentous structure when GDP-tubulin or GTP-Mg-tubulin were used. By HPLC measurements, we demonstrated that a dissociated GTP hydrolysis and exchange of nucleotide occurred during the isomer-induced abnormal assembly. Both isomers inhibited the Mg2+-induced tubulin self-association leading to 42 S double ring formation from GTP-Mg-tubulin or GDP-tubulin. Measurement of their binding under nonassociation conditions revealed a 3-fold decrease in the apparent equilibrium binding constant of the R-isomer to GDP-tubulin relative to GTP-Mg-tubulin. For the S-isomer, the decrease in the binding constant was less pronounced. Binding data, analyzed in terms of a system of linked conformational and association equilibria, provide evidence that the active ("straight") rather than the inactive ("curved") conformation of tubulin differentially recognizes these ligands. Whereas binding of colchicine to tubulin is well-known to induce GTP hydrolysis, this is the first case in which the interaction of a ligand with the colchicine site is shown to be sensitive to the presence of GDP or GTP at the distant nucleotide binding site.

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

“…The apparent binding constants are summarized in Table 1 for comparison. The S isomer bound to tubulin with a slightly higher affinity than the R isomer, as previously shown (43,45). The R and S isomer affinities were not modified in the stathmin complexes.…”

“…For biochemical experiments, tubulin was purified in large scale from lamb brain by ammonium sulfate fractionation, ion exchange chromatography and precipitation with magnesium (33,49). Purified tubulin was stored in liquid nitrogen and prepared for use as described (45). GTP-and GDP-tubulin were prepared as described (45).…”

“…Purified tubulin was stored in liquid nitrogen and prepared for use as described (45). GTP-and GDP-tubulin were prepared as described (45). Tubulin concentration was determined spectrophotometrically at 275 nm in 6 M guanidine hydrochloride (⑀ 275 nm ϭ 109,000 M Ϫ1 ⅐cm Ϫ1 ) or in 0.5% sodium dodecyl sulfate in neutral aqueous buffer (⑀ 275 nm ϭ 107,000…”

“…The binding of R and S isomers induced a decrease of the intrinsic tryptophan tubulin fluorescence (43,45). We used this property to compare the binding affinity of both isomers to the ␣␤-tubulin dimers and to the T 2 Stath complexes.…”

“…Interestingly, the R and S compounds bind to GTP-tubulin with higher affinity than to GDP-tubulin dimers, allosterically distinguishing the bound nucleotide (45). However, allocolchicine (28) and MTC (45) have similar affinities irrespective of the GTP-or GDP-bound nucleotide. The synthetic microtubule inhibitor TN-16 also competes with colchicine for binding to tubulin (46).…”