Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 27, Pages 10166-10173Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0603704103
Keywords
hydrogen/deuterium exchange
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Funding
- NCI NIH HHS [CA 077263, CA 083185, R01 CA083185, R01 CA077263] Funding Source: Medline
- NCRR NIH HHS [S10 RR019352-01, S10 RR019891, S10 RR019352, S10 RR019891-01] Funding Source: Medline
- PHS HHS [DMID-BAA-0338] Funding Source: Medline
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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 dinners and microtubules. The binding of Taxol to beta-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dinners, 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 dinner-dinner 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 dinners.
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