State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China.
Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu 610041, China.
Nat Commun. 2017 Jun 6;8:15787. doi: 10.1038/ncomms15787.
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.
作为细胞骨架的主要组成部分，微管由αβ-微管蛋白异二聚体组成，已被认为是癌症化疗的有吸引力的靶点。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不清楚。在这里，我们报告了一个 2.05Å 的微管与新鉴定的紫杉烷结合位点 MSA 塔卡隆内酯 AJ 的复合物晶体结构。塔卡隆内酯 AJ 与β-微管蛋白 D226 共价结合。在 AJ 结合时，M 环发生构象移位，促进微管蛋白聚合。在这个微管-AJ 复合物中，微管的 E 位被 GTP 占据，而不是 GDP。生化分析证实 AJ 抑制 E 位 GTP 的水解。因此，我们提出 AJ 结合将β-微管蛋白的 E 位锁定在 GTP 优先状态。我们的结果为 MSA 结合与微管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 来克服紫杉烷耐药性。
