Rotational-Echo Double-Resonance NMR Distance Measurements for the Tubulin-Bound Paclitaxel Conformation

J. Am. Chem. Soc., 2007, 129 (2), pp 361--370

ABSTRACT

The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.

The final steps of the synthesis of the target taxol analogue (4) consisted of a coupling reaction between the baccatin core (11) and ¥â-lactam (17) followed by deprotection.

2H{19F} REDOR spectra of tubulin-bound paclitaxel analogue 4 after 64 rotor cycles of dipolar evolution with magic-angle spinning at 8 kHz. The full-echo spectrum is shown in blue, and the dephased spectrum in red. The time domain signals were detected synchronously with the rotor so that all sidebands have been folded into the centerbands. The CD3 peak is assigned zero frequency; the aromatic-D peak appears about 5 ppm to low field. The spectra resulted from the accumulation of 640,000 scans.

2H{19F} REDOR dephasing (DS/S0) for the alanine sample of Figure 2. The experimental dephasing (o) is consistent with that calculated using SIMPSON (solid black line) assuming a single 2H-19F distance of 2.4 A. The corresponding REDOR spectra are shown in the Supporting Information (Figure S2). The inset shows the REDOR dephasing (red) for tubulin-bound paclitaxel analogue 5 after 32 rotor cycles of dipolar evolution with magic-angle spinning at 8 kHz. The REDOR spectra are shown in the Supporting Information (Figure S3). These spectra resulted from the accumulation of 1,056,000 scans. The experimental dephasing is consistent with that calculated using SIMPSON (solid black line) assuming a single 2H3-19F distance of 6.3 A and the 19F chemical shift and 2H quadrupolar tensor parameters of Figure S4 of the Supporting Information. The minor effects of C3 motional averaging were ignored.

Taxane superpositions within the baccatin core. (a) Paclitaxel (magenta), docetaxel (orange), T-Taxol (yellow). (b) As in (a) with PTX-NY in green. The C13 side chain has been truncated at C2' to emphasize differences in the remainder of the molecule.

 

Structures of T-Taxol (yellow) and PTX-NY (green) superposed in the 1JFF tubulin-binding site. (a) The 2Fobsd - Fcalcd omit map is shown as a blue 3D contour. (b) Difference maps (Fobsd - Fcalcd) for the PTX-NY structure. Green corresponds to unfilled density, red to incorrectly filled density.