Microtubules are the target of antitumor drugs such as Taxol. GTP bound to the β subunit of the α/β tubulin heterodimer is hydrolyzed upon assembly of tubulin to microtubules. Taxol induces the assembly of stable microtubules. Although photoaffinity labeling and electron crystallography have localized the binding pocket for Taxol on β-tubulin, they do not provide insight into the mechanism by which Taxol stabilizes microtubules. Hydrogen/Deuterium exchange (HDX) in combination with Liquid Chromatograph-Electrospray ionization mass spectrometry (LC-ESI MS) permits studies of structure and dynamics of biomolecules in solution. A time course of deuterium incorporation was carried out with purified chicken erythrocyte tubulin heterodimers, or microtubules assembled in the presence of GTP or GTP plus Taxol. The global exchange kinetics of tubulin dimers and microtubules was monitored by LC-ESI MS and the deuterium incorporation into tubulin polypeptide chains was calculated by taking into account the deuterium loss during the HPLC step. After 100 minutes of exchange, 251 (430 total exchangeable amide protons) of the amide hydrogens in the tubulin α-chain were replaced by deuterium. The corresponding number for the β-chain was 263 (425 total exchangeable amide protons). After Taxol-induced polymerization, a marked reduction in deuterium incorporation is apparent with only 188 and 178 amide protons in the tubulin α-chain and β-chain, respectively, replaced by deuterium. This protection reflects decreased solvent accessibility or a more rigid conformation in both polypeptide chains. GTP-dependent polymerization reduced the flexibility of microtubules to a lesser extent. The reduced flexibility is preferentially on the β-chain, as suggested by the significantly reduced deuterium incorporation (243 amide protons) on the β-chain, while only slightly reduced deuterium level was observed on the α-chain (248 amide protons). Local HDX on microtubules was analyzed on peptic peptides by LC-MS. As expected, peptides from the Taxol binding pocket were protected against HDX. A comparison of the deuterium incorporation in the presence or absence of GTP- and Taxol allowed us to determine not only the regions involved in Taxol binding, but also the longitudinal and lateral dimer-dimer interactions. The data demonstrate that HDX coupled to mass spectrometry can be used to study microtubule-drug interactions.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]