Structure of tubulin at 6.5 Å and location of the taxol-binding site

Abstract

Tubulin, the major component of microtubules, is a heterodimer of two chains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) and with 40-50% identity. The isotypic variety and conformational flexibility of tubulin have so far made it impossible to obtain crystals for X-ray work. Structural knowledge of tubulin has been limited to about 20 A from X-ray diffraction of oriented microtubules, and from electron microscopy of microtubules and zinc-induced crystalline sheets in negative stain. The sheets consist of protofilaments similar to those in microtubules but associated in an antiparallel arrangement, and their two-dimensional character is ideal for high-resolution electron microscopy. Here we present a three-dimensional reconstruction of tubulin to 6.5 A resolution, obtained by electron crystallography of zinc-induced two-dimensional crystals of the protein. The alpha- and beta-subunits appear topologically similar, in agreement with their sequence homology. Several features can be defined in terms of secondary structure. An apparent alpha-helical portion, adjacent to both interdimer and inter-protofilament contacts, is tentatively attributed to a segment near the carboxy terminus of the protein. We can assign the alpha- and beta-subunits on the basis of projection studies of the binding of taxol, which show one taxol site per tubulin heterodimer, in agreement with the known stoichiometry of taxol in microtubules. These studies indicate that taxol affects the interaction between protofilaments; to our knowledge, this is the first time that a ligand-binding site has been visualized in the tubulin molecule.

Molecular Recognition of Taxol by Microtubules

“…This is supported by the rapid Taxol-induced increase of flexibility of assembled microtubules (28) and by the fast binding and dissociation of 7-O-[N-(4Ј-fluoresceincarbonyl)-L-alanyl]Taxol (Flutax-1) to cellular microtubules (24). All of these characteristics would be compatible with a taxoid binding site in a zone between the protofilaments (20,26,27,29). However, in the model structure of Taxol stabilized microtubules (obtained by fitting into an electron microscopy density map (30) the biochemically well supported (31)(32)(33) high resolution model of the tubulin-Taxol complex (34)), the Taxol binding site is at one side of the ␤-tubulin subunit, clearly facing the microtubular lumen.…”

Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance

“…3,5,7,8). The taxane binding site is on the interior surface of microtubules, resulting in the stabilization of microtubules, increased polymerization, and the suppression of microtubule dynamics, leading to cell cycle arrest in the G 2 /M phase and ultimately, apoptosis (Table 3; refs.…”

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

“…This study focuses on elucidating the structure and interactions of paclitaxel-stabilized MTs (33)(34)(35) assembled at varying Tau/tubulin-dimer molar ratios [Φ Tau = 1/10, 1/13, 1/20, 1/40, and 1/100, spanning the physiological range observed in axons (36)] using synchrotron SAXS in the presence of an osmotic depletant. By varying osmotic pressure [induced by 20k polyethylene oxide (PEO)] (SI Appendix, SI Note S1 and Fig.…”