Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States.
Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.
Elife. 2018 Feb 20;7:e31502. doi: 10.7554/eLife.31502.
Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α units arranged in an α ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α in the presence of both the anticancer drug clofarabine triphosphate and β. Together, these structures support a model for RNR inhibition in which β is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.
核糖核苷酸还原酶（RNR）将核糖核苷酸转化为脱氧核糖核苷酸，这是 DNA 复制和修复所必需的反应。人类 RNR 需要两个亚基才能发挥活性，α亚基包含活性位点，β亚基则含有自由基辅因子。在这里，我们通过低温电子显微镜（EM）呈现了一个 3.3Å 分辨率的人 RNR 与 dATP 抑制状态的结构。该结构是在底物 CDP 和别构调节剂 ATP 和 dATP 的存在下确定的，具有三个排列成α环的α 单元。在接近原子分辨率的水平上，这些数据为 CDP 被别构特异性效应物 dATP/ATP 识别的分子基础提供了深入了解。此外，我们还展示了人 RNR 在存在抗癌药物克拉屈滨三磷酸和β的情况下的较低分辨率 EM 结构。这些结构共同支持了一种 RNR 抑制模型，其中当 α 以稳定的六聚体形式存在时，β 被排除在能够进行自由基转移的结合位置之外。
