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.

中文翻译：

---

他克洛奈德AJ稳定微管的机制。

作为细胞骨架的主要成分，微管由αβ-微管蛋白异二聚体组成，并已被认为是癌症化学疗法的诱人靶标。微管稳定剂（MSA）促进微管蛋白的聚合并稳定聚合物，防止解聚。MSA稳定微管的分子机制仍然难以捉摸。在这里，我们报告了新确定的紫杉烷类MSA与他克洛奈德AJ复合的微管蛋白的2.05Å晶体结构。他克洛奈利AJ与β-微管蛋白D226共价结合。在AJ结合后，M环发生构象变化，以促进微管蛋白聚合。在这个微管蛋白-AJ复合体中，微管蛋白的E部位被GTP占据，而不是被GDP占据。生化分析证实，AJ抑制了E-site GTP的水解。因此，我们建议通过AJ结合将β-微管蛋白E位点锁定为GTP优先状态。我们的结果为MSA结合与微管蛋白核苷酸状态之间的联系提供了实验证据，并将有助于设计新的MSA以克服紫杉烷抗性。