Insights into the mechanism of microtubule stabilization by Taxol.
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.
Xiao
Hui
H
*Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461.
Verdier-Pinard
Pascal
P
Departments of Molecular Pharmacology.|Obstetrics & Gynecology and Women's Health, and.
Fernandez-Fuentes
Narcis
N
Biochemistry, and.
Burd
Berta
B
Departments of Molecular Pharmacology.
Angeletti
Ruth
R
*Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461.
Fiser
Andras
A
Biochemistry, and.
Horwitz
Susan Band
SB
Departments of Molecular Pharmacology, shorwitz@aecom.yu.edu.
Orr
George A
GA
Departments of Molecular Pharmacology.
D000818:Animals; D002645:Chickens; D041961:Deuterium Exchange Measurement; D019281:Dimerization; D008870:Microtubules / Q000737:chemistry* / Q000187:drug effects*; D008958:Models, Molecular; D017239:Paclitaxel / Q000737:chemistry / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D014404:Tubulin; D017239:Paclitaxel