着丝粒是存在于人类染色体着丝粒的多蛋白质组装体，在细胞有丝分裂中发挥着至关重要的作用。 CENP-T 和 CENP-W 链形成异二聚体，它是内部着丝粒的组成部分，一侧与接头 DNA 相互作用，另一侧与外着丝粒相互作用。此外，CENP-T-W 二聚体与参与形成内部动粒的其他调节蛋白相互作用。 CENP-T-W 二聚体形成过程中蛋白质-蛋白质相互作用 (PPI) 界面处 CENP-W 中不同氨基酸的具体作用仍不完全清楚。由于细胞分裂在癌症等疾病中会出现错误，这种 CENP-T-W 合作伙伴关系是可以恢复健康细胞分裂的新药的潜在目标。我们采用分子对接、结合自由能计算和分子动力学 (MD) 模拟来研究 CENP-W 链中氨基酸取代对 CENP-T-W 二聚体形成的破坏性影响。通过进行分子对接研究和分析氢键相互作用，我们鉴定了 CENP-W 中的关键残基（ASN-46、ARG-53、LEU-83、SER-86、ARG-87 和 GLY-88）以供进一步研究。通过定点诱变和随后的结合自由能计算，我们改进了突变体的选择。我们选择了 CENP-W 的四个突变体（N46K、R53K、L83K 和 R87E）来评估它们形成 CENP-T-W 二聚体的比较潜力。我们对 250 ns 长的分析表明，用 LYS 替换 CENP-W 中的 LEU83 和 ARG53 残基会显着破坏 CENP-T-W 二聚体的形成。总之，LEU83 和 ARG53 在细胞有丝分裂最终所需的 CENP-T 和 CENP-W 二聚化中发挥关键作用。我们的研究结果不仅加深了我们对细胞分裂的理解，还暗示了令人兴奋的药物靶点可能性。© 2023 作者。 《细胞生物化学杂志》由 Wiley periodicals LLC 出版。

Kinetochores are multi-protein assemblies present at the centromere of the human chromosome and play a crucial role in cellular mitosis. The CENP-T and CENP-W chains form a heterodimer, which is an integral part of the inner kinetochore, interacting with the linker DNA on one side and the outer kinetochore on the other. Additionally, the CENP-T-W dimer interacts with other regulatory proteins involved in forming inner kinetochores. The specific roles of different amino acids in the CENP-W at the protein-protein interaction (PPI) interface during the CENP-T-W dimer formation remain incompletely understood. Since cell division goes awry in diseases like cancer, this CENP-T-W partnership is a potential target for new drugs that could restore healthy cell division. We employed molecular docking, binding free energy calculations, and molecular dynamics (MD) simulations to investigate the disruptive effects of amino acids substitutions in the CENP-W chain on CENP-T-W dimer formation. By conducting a molecular docking study and analysing hydrogen bonding interactions, we identified key residues in CENP-W (ASN-46, ARG-53, LEU-83, SER-86, ARG-87, and GLY-88) for further investigation. Through site-directed mutagenesis and subsequent binding free energy calculations, we refined the selection of mutant. We chose four mutants (N46K, R53K, L83K, and R87E) of CENP-W to assess their comparative potential in forming CENP-T-W dimer. Our analysis from 250 ns long revealed that the substitution of LEU83 and ARG53 residues in CENP-W with the LYS significantly disrupts the formation of CENP-T-W dimer. In conclusion, LEU83 and ARG53 play a critical role in CENP-T and CENP-W dimerization which is ultimately required for cellular mitosis. Our findings not only deepen our understanding of cell division but also hint at exciting drug-target possibilities.© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.