Disruption of an oligomeric interface prevents allosteric inhibition of class Ia ribonucleotide reductase.

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α subunit that houses the active site and allosteric regulatory sites and a β subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact αβ state allowing for radical transfer from β to α RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied class Ia RNR, dATP binding to an allosteric site on α promotes formation of an αβ ring-like state. Here, we investigate whether the αβ formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α, S39R-α, S39F-α, E42K-α, or L43Q-α) that disrupt the αβ oligomer abrogate dATP-mediated inhibition, consistent with the idea that αβ formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-β interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-β interface whose substitution has previously been shown to cause a mutator phenotype in ( S39F-α or E42K-α) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.