How ATP and\ndATP Act as Molecular Switches to Regulate\nEnzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide\nReductase

Class Ia ribonucleotide\nreductases (RNRs) are allosterically\nregulated\nby ATP and dATP to maintain the appropriate deoxyribonucleotide levels\ninside the cell for DNA biosynthesis and repair. RNR activity requires\nprecise positioning of the β<sub>2</sub> and α<sub>2</sub> subunits for the transfer of a catalytically essential radical species.\nExcess dATP inhibits RNR through the creation of an α–β\ninterface that restricts the ability of β<sub>2</sub> to obtain\na position that is capable of radical transfer. ATP breaks the α–β\ninterface, freeing β<sub>2</sub> and restoring enzyme activity.\nHere, we investigate the molecular basis for allosteric activity regulation\nin the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures\nand accompanying biochemical and mutagenesis studies. We find that\nwhen dATP is bound to the N-terminal regulatory cone domain in α,\na helix unwinds, creating a binding surface for β. When ATP\ndisplaces dATP, the helix rewinds, dismantling the α–β\ninterface. This reversal of enzyme inhibition requires that two ATP\nmolecules are bound in the cone domain: one in the canonical nucleotide-binding\nsite (site 1) and one in a site (site 2) that is blocked by phenylalanine-87\nand tryptophan-28 unless ATP is bound in site 1. When ATP binds to\nsite 1, histidine-59 rearranges, prompting the movement of phenylalanine-87\nand trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59,\npreventing its movement. The importance of site 2 in the restoration\nof RNR activity by ATP is confirmed by mutagenesis. These findings\nhave implications for the design of bacterial RNR inhibitors.