3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.

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.