Insights into the mechanism of microtubule stabilization by Taxol Export

@article{citeulike:4680423, abstract = {10.1073/pnas.0603704103 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.}, author = {Xiao, Hui and Verdier-Pinard, Pascal and Fernandez-Fuentes, Narcis and Burd, Berta and Angeletti, Ruth and Fiser, Andras and Horwitz, Susan B. and Orr, George A.}, citeulike-article-id = {4680423}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0603704103}, citeulike-linkout-1 = {http://www.pnas.org/content/103/27/10166.abstract}, citeulike-linkout-2 = {http://www.pnas.org/content/103/27/10166.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/16801540}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=16801540}, doi = {10.1073/pnas.0603704103}, journal = {Proceedings of the National Academy of Sciences}, keywords = {chapter}, month = {July}, number = {27}, pages = {10166--10173}, posted-at = {2009-05-29 17:36:30}, priority = {2}, title = {Insights into the mechanism of microtubule stabilization by Taxol}, url = {http://dx.doi.org/10.1073/pnas.0603704103}, volume = {103}, year = {2006} }

TY - JOUR ID - citeulike:4680423 L3 - citeulike-article-id:4680423 N2 - 10.1073/pnas.0603704103 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. IS - 27 JF - Proceedings of the National Academy of Sciences EP - 10173 TI - Insights into the mechanism of microtubule stabilization by Taxol VL - 103 SP - 10166 KW - chapter AU - Xiao, Hui AU - Verdier-Pinard, Pascal AU - Fernandez-Fuentes, Narcis AU - Burd, Berta AU - Angeletti, Ruth AU - Fiser, Andras AU - Horwitz, Susan AU - Orr, George PY - 2006/07/05/ UR - http://dx.doi.org/10.1073/pnas.0603704103 ER -