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(2) units arranged in an α(6) 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(6) in the presence of both the anticancer drug clofarabine triphosphate and β(2). Together, these structures support a model for RNR inhibition in which β(2) is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.