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 ␣͞␤-tubulin heterodimer, GTP bound to ␤-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to ␤-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 ␤-and ␣-tubulin upon microtubule stabilization by Taxol. Hydrogen͞deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both ␤-and ␣-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.hydrogen͞deuterium exchange

Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

“…In addition, our 2ME2-resistant leukemia cells acquired two novel mutations in class I h-tubulin, S25N and A248T, which were confirmed at the protein level by MS analysis. The S25N mutation is at the NH 2 terminus of h-tubulin (28) and forms part of the paclitaxel-binding pocket (29). Furthermore, this mutation is close to the htubulin M-loop, which forms major lateral interactions in microtubule assembly (30).…”

Probing the origin of tubulin rigidity with molecular simulations

“…Experiments found that, in vivo, such a delicate balance is maintained because of a variety of factors that modulate the dynamic instability of MTs: GTP hydrolysis (3,9), molecular motors such as Kin-13 and other microtubuleassociated proteins (MAPs), and cofactors such as E and B, which increase the depolymerization rate (10,11); MAPs such as CLIP-170, which stabilize the MT lattice preventing catastrophes (12); and katanin and spastin, which sever MTs (13). However, in vitro MT dynamic instability occurs under modest conditions: either as a result of GTP hydrolysis (14) or under cold denaturation even in a nonhydrolyzable GMPCPP MT variant (9).…”

“…Because of their dynamic instability, i.e., stochastic alternation of slow growth and rapid shrinking phases between MTs and soluble tubulin subunits, MTs are able to transport chromosomes and other cellular organelles inside the cell (1). Blockage of dynamic instability by drugs such as taxol, which reduce the flexibility of the MT structure, promotes mitotic arrest and leads to cell death (2,3). This behavior makes MTs a main target for cancer drugs (2), but efforts to design effective drugs are hampered, in part, by the lack of clear understanding of the microscopic origin of MT instability and of the MT behavior under tension (4).…”

Profile of Susan Band Horwitz

“…Deuterium incorporation of the microtubules was then measured, providing a picture of the conformational changes between free tubulin and Taxolbound tubulin. Taxol was found to modify tubulin conformation not just at the binding sites but at allosteric sites as well (1). The results indicated that, whereas in the absence of Taxol the tubulin dimers can adopt a curved conformation associated with the destabilization of microtubules, in the drug's presence the dimer is locked into a straight conformation promoting assembly of microtubules and stabilization.…”