Changes in Microtubule Protofilament Number Induced by Taxol Binding to an Easily Accessible Site

Abstract

We have investigated the accessibility of the Taxolbinding 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 ϳ0.5 min, employing GDP-tubulintaxoid microtubules. Correspondingly, synchrotron xray 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 ϳ1 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.

Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thalianamicrotubule protofilament model and in the cells

“…When taxol was excluded during preparation of unfixed samples, staining of microtubules with 18D6 antibody was reduced (Additional file 2). An altering effect of taxol on the arrangement of tubulin protofilaments was reported [27,28]. We thus propose that the exposure of 18D6 epitope on unfixed A. thaliana microtubules might be caused by conformation changes generated by taxol used for stabilization of microtubules.…”

Mechanism of Electromechanical Coupling in Voltage-Gated Potassium Channels

“…Although the gating mechanism is similar to other Kv channels (Diaz et al, 1998; Cui and Aldrich, 2000; Ma et al, 2006; Savalli et al, 2006), differences to the delayed rectifier Kv channels have been reported (Li and Aldrich, 2004, 2006; Zhou et al, 2011). The positive S4 residues do not as dominantly control the voltage dependence; instead charged residues throughout the voltage sensor domain lead to more global conformational changes (Ma et al, 2006; Savalli et al, 2006; Pantazis et al, 2009, 2010).…”

Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: Implications for chemotherapy-induced peripheral neuropathy

“…The differences may be due in part to the higher binding affinity of ixabepilone relative to paclitaxel (Perez, 2009), but they may also arise from different effects on microtubule structure. For example, paclitaxel and the epothilones have drug-specific effects on microtubule protofilament number (Diaz et al, 1998; Meurer-Grob et al, 2001). In addition, both drugs also induce conformational changes in the carboxy terminus of tubulin (Khrapunovich-Baine et al, 2011), a region that plays a role in the binding of motor proteins and other MAPs (Khrapunovich-Baine et al, 2009, 2011) and in the regulation of kinesin-1 ATPase activity (Uchimura et al, 2010).…”