Understanding tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin

Abstract

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

Fig. 1.

Structure of Taxol (paclitaxel).

Fig. 2.

Mutated yeast tubulin displays Taxol and epothilone-related activity. (A) Assembly of mutated yeast tubulin in the presence of Taxol (○) and epothilone B (▿) and of wild-type yeast (▪) and bovine brain (•) tubulin in the presence of Taxol. The reactions contained 5 μM (0.5 mg/ml) mutated tubulin from the yeast strain MGY1-tax, wild-type yeast tubulin, or bovine brain tubulin and 2.5% DMSO (see Materials and Methods). They were incubated for 30 min at 30°C under conditions that do not promote assembly in the absence of Taxol or epothilone B. The amount of polymer was determined by sedimentation assay. (B) Electron micrograph of negatively stained microtubules formed from mutated yeast tubulin in the presence of 3 μM Taxol. Reaction conditions were as described for A. (Bar = 100 nm.)

Fig. 3.

Stoichiometry and competition by epothilone B of Taxol binding to polymerized, mutated yeast tubulin. (A) Stoichiometry of Taxol binding. (B) Inhibition of Taxol binding by epothilone B. Mutated yeast tubulin was purified from the yeast strain MGY1-tax. The reactions were performed as described for Fig. 2 A. The ratio of Taxol to tubulin was determined from the amount of [³H]Taxol bound to the tubulin polymer after sedimentation. For B, the reactions contained 3 μM [³H]Taxol, with a total of 5% DMSO.

Fig. 4.

Taxol binding site on mammalian β-tubulin. The location of residues Lys-19, Val-23, Asp-26, His-227, and Phe-270 are indicated and are shown in dark gray. Labels on Taxol (gray) denote the following: I, C3′ phenyl ring; II, C3′ benzamido phenyl ring; and III, C2 benzoyl phenyl ring. Specific regions of β-tubulin that form the binding pocket are labeled, including α-helices H1, H7, H9, and H10, the β-strands B7–B10, and the B7–H9 M-loop. The structure was drawn with the modeling programs molscript (34) and raster3d (35) by using the coordinates (PDB ID code 1JFF) determined by Snyder et al. (16) for T-Taxol bound to the refined model of bovine brain tubulin (36).