Department of Biochemistry, University of Cambridge, Cambridge, UK.
Heart and Lung Research Institute, Department of Medicine, University of Cambridge, Royal Papworth Hospital NHS Foundation Trust, Cambridge, UK.
Nat Struct Mol Biol. 2023 Feb;30(2):140-147. doi: 10.1038/s41594-022-00881-w. Epub 2023 Jan 5.
DNA-dependent protein kinase (DNA-PK), a multicomponent complex including the DNA-PK catalytic subunit and Ku70/80 heterodimer together with DNA, is central to human DNA damage response and repair. Using a DNA-PK-selective inhibitor (M3814), we identified from one dataset two cryo-EM structures of the human DNA-PK complex in different states, the intermediate state and the active state. Here we show that activation of the kinase is regulated through conformational changes caused by the binding ligand and the string region (residues 802-846) of the DNA-PK catalytic subunit, particularly the helix-hairpin-helix motif (residues 816-836) that interacts with DNA. These observations demonstrate the regulatory role of the ligand and explain why DNA-PK is DNA dependent. Cooperation and coordination among binding partners, disordered flexible regions and mechanically flexible HEAT repeats modulate the activation of the kinase. Together with previous findings, these results provide a better molecular understanding of DNA-PK catalysis.
DNA 依赖性蛋白激酶 (DNA-PK) 是一种多组分复合物，包括 DNA-PK 催化亚基和 Ku70/80 异源二聚体以及 DNA，是人类 DNA 损伤反应和修复的核心。我们使用 DNA-PK 选择性抑制剂 (M3814)，从一个数据集鉴定出两种不同状态下的人 DNA-PK 复合物的低温电镜结构，即中间状态和活性状态。在这里，我们表明激酶的激活是通过配体结合和 DNA-PK 催化亚基的串扰区（残基 802-846）引起的构象变化来调节的，特别是与 DNA 相互作用的螺旋-发夹-螺旋模体（残基 816-836）。这些观察结果表明了配体的调节作用，并解释了为什么 DNA-PK 是 DNA 依赖性的。结合伴侣、无规则柔性区域和机械柔性 HEAT 重复的合作和协调调节激酶的激活。结合以前的发现，这些结果提供了对 DNA-PK 催化的更好的分子理解。
