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 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.
authors.affiliation: *Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Hui
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Departments of Molecular Pharmacology.|Obstetrics & Gynecology and Women's Health, and.
authors.forename: Pascal
authors.initials: P
authors.lastname: Verdier-Pinard
authors.affiliation: Biochemistry, and.
authors.forename: Narcis
authors.initials: N
authors.lastname: Fernandez-Fuentes
authors.affiliation: Departments of Molecular Pharmacology.
authors.forename: Berta
authors.initials: B
authors.lastname: Burd
authors.affiliation: *Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Ruth
authors.initials: R
authors.lastname: Angeletti
authors.affiliation: Biochemistry, and.
authors.forename: Andras
authors.initials: A
authors.lastname: Fiser
authors.affiliation: Departments of Molecular Pharmacology, shorwitz@aecom.yu.edu.
authors.forename: Susan Band
authors.initials: SB
authors.lastname: Horwitz
authors.affiliation: Departments of Molecular Pharmacology.
authors.forename: George A
authors.initials: GA
authors.lastname: Orr
mesh_terms: 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
chemical_list: D010446:Peptide Fragments; D014404:Tubulin; D017239:Paclitaxel