Changes in microtubule protofilament number induced by Taxol binding to an easily accessible site. Internal microtubule dynamics

Abstract

We have investigated the accessibility of the Taxol-binding site and the effects of Taxol binding on the structures of assembled microtubules. Taxol and docetaxel readily bind to and dissociate from microtubules, reaching 95% ligand exchange equilibrium in less than 3 min under our solution conditions (microtubules were previously assembled from GTP-tubulin, GTP-tubulin and microtubule-associated proteins, or GDP-tubulin and taxoid). Microtubules assembled from purified tubulin with Taxol are known to have typically one protofilament less than with the analogue docetaxel and control microtubules. Surprisingly, Taxol binding and exchange induce changes in the structure of preformed microtubules in a relatively short time scale. Cryoelectron microscopy shows changes toward the protofilament number distribution characteristic of Taxol or docetaxel, with a half-time of approximately 0.5 min, employing GDP-tubulin-taxoid microtubules. Correspondingly, synchrotron x-ray solution scattering shows a reduction in the mean microtubule diameter upon Taxol binding to microtubules assembled from GTP-tubulin in glycerol-containing buffer, with a structural relaxation half-time of approximately 1 min. These results imply that microtubules can exchange protofilaments upon Taxol binding, due to internal dynamics along the microtubule wall. The simplest interpretation of the relatively fast taxoid exchange observed and labeling of cellular microtubules with fluorescent taxoids, is that the Taxol-binding site is at the outer microtubule surface. On the contrary, if Taxol binds at the microtubule lumen in agreement with the electron crystallographic structure of tubulin dimers, our results suggest that the inside of microtubules is easily accessible from the outer solution. Large pores or moving lattice defects in microtubules might facilitate the binding of taxoids, as well as of possible endogenous cellular ligands of the inner microtubule wall.