Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Nat Struct Mol Biol. 2024 Jun;31(6):861-873. doi: 10.1038/s41594-024-01230-9. Epub 2024 Mar 8.
Biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell's chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. One of the outer layer main components is a ten-subunit assembly comprising Knl1C, Mis12C and Ndc80C (KMN) subcomplexes. The KMN is highly elongated and docks on kinetochores and microtubules through interfaces at its opposite extremes. Here, we combine cryogenic electron microscopy reconstructions and AlphaFold2 predictions to generate a model of the human KMN that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments, we demonstrate that this mutual organization strongly stabilizes the KMN assembly. Our work thus reports a comprehensive structural and functional analysis of this part of the kinetochore microtubule-binding machinery and elucidates the path of connections from the chromatin-bound components to the force-generating components.
在细胞分裂过程中，染色体的正确定向对于精确分配母细胞的染色体到两个子细胞中是必要的。动粒是在称为着丝粒的专门染色体位置上构建的大型分层结构，通过结合和感知纺锤体微管来促进正确定向。其外层的主要成分之一是一个由 Knl1C、Mis12C 和 Ndc80C（KMN）亚基组成的十亚基组装体。KMN 非常细长，通过其相对极端的界面连接在动粒和微管上。在这里，我们结合低温电子显微镜重建和 AlphaFold2 预测，生成了一个人类 KMN 的模型，揭示了所有的 KMN 内部界面。我们确定并功能验证了将 Mis12C 连接到 Ndc80C 和 Knl1C 的两个相互作用界面。通过靶向干扰实验，我们证明了这种相互组织强烈稳定了 KMN 组装体。因此，我们的工作报告了对这个动粒微管结合机制的一部分的全面结构和功能分析，并阐明了从染色质结合成分到产生力的成分的连接路径。
