Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli 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 α2 subunit that houses the active site and allosteric regulatory sites and a β2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2 β2 state allowing for radical transfer from β2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4 β4 ring-like state. Here, we investigate whether the α4 β4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α2 , S39R-α2 , S39F-α2 , E42K-α2 , or L43Q-α2 ) that disrupt the α4 β4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4 β4 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 Escherichia coli ( i.e. S39F-α2 or E42K-α2 ) 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.