Mechanism of microtubule stabilization by taccalonolide AJ

Abstract

As a major component of the cytoskeleton, microtubules consist of αβ-tubulin heterodimers and have been recognized as attractive targets for cancer chemotherapy. Microtubule-stabilizing agents (MSAs) promote polymerization of tubulin and stabilize the polymer, preventing depolymerization. The molecular mechanisms by which MSAs stabilize microtubules remain elusive. Here we report a 2.05 Å crystal structure of tubulin complexed with taccalonolide AJ, a newly identified taxane-site MSA. Taccalonolide AJ covalently binds to β-tubulin D226. On AJ binding, the M-loop undergoes a conformational shift to facilitate tubulin polymerization. In this tubulin–AJ complex, the E-site of tubulin is occupied by GTP rather than GDP. Biochemical analyses confirm that AJ inhibits the hydrolysis of the E-site GTP. Thus, we propose that the β-tubulin E-site is locked into a GTP-preferred status by AJ binding. Our results provide experimental evidence for the connection between MSA binding and tubulin nucleotide state, and will help design new MSAs to overcome taxane resistance.

Elucidating target specificity of the taccalonolide covalent microtubule stabilizers employing a combinatorial chemical approach

“…6e). These results confirm the critical role of the 22,23-epoxide and β-tubulin D226 in taccalonolide-tubulin binding 18 . Encouraged by the results of the D226 mutagenesis, additional β-tubulin mutants were constructed in a similar fashion.…”

“…While we were exploring strategies for generating an optimal taccalonolide-based chemical probe, Wang et al reported the crystal structure (PDB ID: 5EZY) of tubulin complexed with taccalonolide AJ (2) (Fig. 1a, c) 18 . The authors proposed an unusual reaction mechanism intending to explain the covalent bond formation between the 22,23-epoxy moiety of 2 and βtubulin D226.…”

“…The generation of the potent fluorogenic taccalonolide probe Flu-tacca-7 (11) provides the opportunity to evaluate the covalent binding specificity of the taccalonolides in cell-based assays and fully define the key structural elements of both taccalonolides and β-tubulin that are essential for drug binding. The 22,23-epoxide moiety has been suggested to be critical for the antiproliferative and microtubule stabilizing effects of the taccalonolides 18,24,33 . Thus, we compared the antiproliferative activities, cellular localization, and proteome reactivity profiles of Flu-tacca-7 (11) and its 22,23-ene analogue 10.…”

“…However, a major limitation of the taxanes is acquired drug resistance. The taccalonolides are a class of microtubule stabilizers that covalently bind β-tubulin 17,18 and effectively circumvent clinically relevant models of resistance to taxanes both in vitro and in vivo [19][20][21] . Despite their promising therapeutic potential, the covalent nature of taccalonolide binding has hampered our ability to perform detailed binding studies using conventional approaches 22,23 .…”

Molecular modeling study on the differential microtubule‐stabilizing effect in singly‐ and doubly‐bonded complexes with peloruside A and paclitaxel

“…The helical M‐loop conformation in the ligand‐free and TX‐bonded states was early reported by Alushin et al and has also been observed in other TX‐site binders as the MT‐stabilizing agent zampanolide . Such conformational change has been proposed to facilitate the lateral interaction between PFs in MTs . In the case of the doubly‐bonded system, the helical conformation of the M‐loop was retained and the loss of secondary structure induced by the individual association of PLA was overcome, thus suggesting that the M‐loop conformation is mostly modulated by TX‐site binders.…”