Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC50 values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.

中文翻译：

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结合cryptophycin-52 后微管蛋白的构象变化揭示了其作用机制。

Cryptophycin-52 (Cp-52) 可能是已知的最有效的抗癌药物，其 IC50 值在低皮摩尔范围内，但其在微管蛋白上的结合位点和作用机制尚不清楚。在这里，我们通过冷冻电镜确定了 Cp-52 及其母体化合物cryptophycin-1 在 HeLa 微管蛋白上的结合位点，分辨率分别为 3.3 Å 和 3.4 Å，并通过分子动力学进一步表征了这种结合模拟。结合位点被确定为位于微管蛋白二聚体界面，并与另一种细胞毒性微管蛋白抑制剂美登素部分重叠。结合在与微管晶格不相容的微管蛋白二聚体内部和之间诱导弯曲。构象变化发生在 α-微管蛋白和 β-微管蛋白中，特别是在 H8 和 H10 螺旋中，α 和 β 单体之间以及 Cp-52 结合和隐霉素 1 结合的微管蛋白之间存在明显差异。根据这些结果，我们已经确定：（i）抑制微管聚合和解聚的作用机制，（ii）Cp-52 对微管蛋白的亲和力如何增强，以及（iii）用于靶向递送的接头在哪里可以最佳地附着在该分子上。