Insights into the mechanism of microtubule stabilization by Taxol

Abstract

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the alpha/beta-tubulin heterodimer, GTP bound to beta-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to beta-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both beta- and alpha-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both beta-and alpha-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer-dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography-electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.

Fig. 1.

Ratios of percentages of HDX in peptic peptides along the sequence of tubulin. The bar diagrams show relative HDX in the three systems studied: tubulin dimer (DIMER), TX-MT, and microtubules polymerized in the presence of GTP alone (GTP-MT). Ratios of HDX percentages of peptic peptides at 35 min from α-tubulin (A) and from β-tubulin (B) are represented for DIMER to TX-MT as red bars, DIMER to GTP-MT as blue bars, and GTP-MT to TX-MT as green bars. The first and last residues of each peptide are indicated on the left axis from the N terminus at the top to the C terminus. Ratio values are indicated on the top horizontal axis. The bottom colored bar indicates the color-coding of the HDX ratio (r) that is used for the models in Figs. 2 and 3 (dark gray, r ≤ 1.2; yellow, 1.2 < r ≤ 2.0; orange, 2.0 < r ≤ 3.0; red, r > 3).

Fig. 2.

Mapping HDX ratio changes on the tubulin model. (A) DIMER to TX-MT. (B) DIMER to GTP-MT. (C) GTP-MT to TX-MT. (Upper) Tubulin structure as ribbons. (Lower) Molecular surface of tubulin. Peptic peptides are colored according to the code indicated in Fig. 1, and light gray areas are due to missing peptides.

Fig. 3.

Mapping of the HDX ratio, GTP-MT to TX-MT, on a microtubule model. The extent of protection against HDX is color-coded as in Figs. 1 and 2. (A) View of a portion of two adjacent protofilaments from inside to outside in a microtubule. The right protofilament is facing the viewer, whereas the left one is coming toward the viewer. (B) View of a portion of two adjacent protofilaments from outside to inside of a microtubule. The left protofilament is facing the viewer, whereas the right one is going away from the viewer. (C) Lateral interfaces of dimers. (D) Intradimer interface with β-tubulin at the top and α-tubulin at the bottom. (E) Interdimer interfaces in protofilaments. (Upper) View of protofilaments in B from the top (β-tubulin). (Lower) View of protofilaments in B from the bottom (α-tubulin). Lateral relative orientation of dimers is based on a TX-MT made of 12-protofilaments. Taxol is in blue, and GDP is in green. The surfaces were superimposed by transparency on the ribbon structures. Some of the peptides that are discussed in the text are numbered, and their location is indicated on the different views of the HDX map.

Fig. 4.

Diagrams of HDX results at the interfaces of tubulin dimers in microtubules. (A) Diagram of HDX ratio, DIMER to GTP-MT, with stathmin (green circles) bound to the outside of the microtubule. The lateral contacts are represented as gray discs; longitudinal contacts and H10 at the inter- and intradimer, respectively, are represented as yellow discs. Depolymerizing outward forces are represented by white dashed arrows. (B) Diagram of HDX ratio GTP-MT to TX-MT with Taxol (blue discs) bound to the inside of the microtubule. Lateral contacts and H10 are represented as yellow and red discs, respectively. Peptide β167–187 is represented as orange ovals, and peptides α253–271 and α344–351 are represented as red ovals, at the top of β-tubulin and at the bottom of α-tubulin, respectively. Longitudinal, lateral, and resultant forces induced by Taxol stabilization are represented by dashed white arrows. Superimposition of the top of β-tubulin with the bottom of α-tubulin is represented by dashed ellipses. For simplification and to help visualize the superimposition of GTP and Taxol contributions to the establishment of longitudinal and lateral contacts, the microtubule in A is made only of 12-protofilaments as in B where Taxol is present. Distances between interfaces in B have been slightly increased to represent the dimer longitudinal expansion induced by Taxol. ①, top of β-tubulin plus lateral contact; ②, intradimer interface; ③, bottom of α-tubulin plus interdimer interface.