Insights into the mechanism of microtubule stabilization by Taxol

Hui Xiao, Pascal Verdier-Pinard, Narcis Fernandez-Fuentes, Berta Burd, Ruth Angeletti, Andras Fiser, Susan Band Horwitz, George A. Orr

Research output: Contribution to journalArticle

184 Scopus citations

Abstract

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the α/β-tubulin heterodimer, GTP bound to β-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to β-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 β- and α-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 β-and α-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.

Original languageEnglish (US)
Pages (from-to)10166-10173
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number27
DOIs
StatePublished - Jul 4 2006

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Keywords

  • Deuterium exchange
  • Hydrogen

ASJC Scopus subject areas

  • General

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