Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Nucleic Acids Res. 2019 Jan 10;47(1):468-479. doi: 10.1093/nar/gky1108.
The kinetochore is a proteinaceous complex that is essential for proper chromosome segregation. As a core member of the inner kinetochore, defects of each subunit in the CENP-H/I/K complex cause dysfunction of kinetochore that leads to chromosome mis-segregation and cell death. However, how the CENP-H/I/K complex assembles and promotes kinetochore function are poorly understood. We here determined the crystal structures of CENP-I N-terminus alone from Chaetomium thermophilum and its complex with CENP-H/K from Thielavia terrestris, and verified the identified interactions. The structures and biochemical analyses show that CENP-H and CENP-K form a heterodimer through both N- and C-terminal interactions. CENP-I integrates into the CENP-H/K complex by binding to the C-terminus of CENP-H, leading to formation of the ternary complex in which CENP-H is sandwiched between CENP-K and CENP-I. Our sequence comparisons and mutational analyses showed that this architecture of the CENP-H/I/K complex is conserved in human. Mutating the binding interfaces of CENP-H for either CENP-K or CENP-I significantly reduced their localizations at centromeres and induced massive chromosome alignment defects during mitosis, suggesting that the identified interactions are critical for CENP-H/I/K complex assembly at the centromere and kinetochore function. Altogether, our findings unveil the evolutionarily conserved assembly mechanism of the CENP-H/I/K complex that is critical for proper chromosome alignment.
着丝粒是一种对于染色体正确分离至关重要的蛋白复合物。作为着丝粒内层复合物的核心成员，CENP-H/I/K 复合物中每个亚基的缺陷都会导致着丝粒功能障碍，从而导致染色体错误分离和细胞死亡。然而，CENP-H/I/K 复合物是如何组装并促进着丝粒功能的，目前仍知之甚少。我们在这里确定了来自嗜热毛壳菌的 CENP-I N 端以及与其来自土曲霉的 CENP-H/K 的复合物的晶体结构，并验证了鉴定出的相互作用。结构和生化分析表明，CENP-H 和 CENP-K 通过 N 端和 C 端相互作用形成异二聚体。CENP-I 通过与 CENP-H 的 C 端结合整合到 CENP-H/K 复合物中，导致形成三元复合物，其中 CENP-H 夹在 CENP-K 和 CENP-I 之间。我们的序列比较和突变分析表明，该 CENP-H/I/K 复合物的结构在人类中是保守的。突变 CENP-H 与 CENP-K 或 CENP-I 的结合界面显著降低了它们在着丝粒处的定位，并在有丝分裂过程中诱导大量染色体对齐缺陷，这表明鉴定出的相互作用对于 CENP-H/I/K 复合物在着丝粒处的组装和着丝粒功能至关重要。总之，我们的发现揭示了 CENP-H/I/K 复合物的进化保守组装机制，该机制对于正确的染色体排列至关重要。
