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

177 Citations (Scopus)

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

Fingerprint

Tubulin
Paclitaxel
Microtubules
Deuterium
Guanosine Triphosphate
Hydrogen
Liquid Chromatography
Protein Binding
Polymerization
Antineoplastic Agents
Digestion
Polymers
Cell Death
Binding Sites
Ligands
Peptides

Keywords

  • Deuterium exchange
  • Hydrogen

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Insights into the mechanism of microtubule stabilization by Taxol. / Xiao, Hui; Verdier-Pinard, Pascal; Fernandez-Fuentes, Narcis; Burd, Berta; Angeletti, Ruth; Fiser, Andras; Band Horwitz, Susan; Orr, George A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 27, 04.07.2006, p. 10166-10173.

Research output: Contribution to journalArticle

Xiao, Hui ; Verdier-Pinard, Pascal ; Fernandez-Fuentes, Narcis ; Burd, Berta ; Angeletti, Ruth ; Fiser, Andras ; Band Horwitz, Susan ; Orr, George A. / Insights into the mechanism of microtubule stabilization by Taxol. In: Proceedings of the National Academy of Sciences of the United States of America. 2006 ; Vol. 103, No. 27. pp. 10166-10173.
@article{063b5af75b2a40ea89ece33b3633f0ee,
title = "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 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.",
keywords = "Deuterium exchange, Hydrogen",
author = "Hui Xiao and Pascal Verdier-Pinard and Narcis Fernandez-Fuentes and Berta Burd and Ruth Angeletti and Andras Fiser and {Band Horwitz}, Susan and Orr, {George A.}",
year = "2006",
month = "7",
day = "4",
doi = "10.1073/pnas.0603704103",
language = "English (US)",
volume = "103",
pages = "10166--10173",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "27",

}

TY - JOUR

T1 - Insights into the mechanism of microtubule stabilization by Taxol

AU - Xiao, Hui

AU - Verdier-Pinard, Pascal

AU - Fernandez-Fuentes, Narcis

AU - Burd, Berta

AU - Angeletti, Ruth

AU - Fiser, Andras

AU - Band Horwitz, Susan

AU - Orr, George A.

PY - 2006/7/4

Y1 - 2006/7/4

N2 - 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.

AB - 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.

KW - Deuterium exchange

KW - Hydrogen

UR - http://www.scopus.com/inward/record.url?scp=33745918018&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33745918018&partnerID=8YFLogxK

U2 - 10.1073/pnas.0603704103

DO - 10.1073/pnas.0603704103

M3 - Article

C2 - 16801540

AN - SCOPUS:33745918018

VL - 103

SP - 10166

EP - 10173

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 27

ER -