Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone allosterically mediates substrate binding to the active site

Abstract

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding ATP or dATP it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerization and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and disordering of the GRD. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind, suggesting that dATP inhibition in anaerobic RNRs acts by disordering of the GRD more than 30 Å away from the dATP molecule, thereby preventing both substrate binding and radical mobilisation. The structures implicate a complex network of activity regulation involving the GRD, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site.