Abstract
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 α2β2 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 α4β4 ring-like state. Here, we investigate whether the α4β4 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 α4β4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4β4 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.
Highlights
Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair
The prototypic enzyme for understanding RNR chemistry and regulation is the class Ia RNR from Escherichia coli. This enzyme consists of two dimeric subunits, ␣2 and 2. ␣2 contains the catalytic machinery for nucleotide reduction as well as two allosteric nucleotide-binding sites: an allosteric specificity site at the dimer interface that modulates the affinity of the enzyme for pyrimidine versus purine substrates [5,6,7] and an allosteric activity site at an N-terminal cone domain that modulates overall activity (Fig. 1B) (8 –10). 2 contains the diferric tyrosyl radical cofactor that is essential to RNR’s catalytic mechanism (Fig. 1B) [11,12,13,14]
DATP binding to the N-terminal cone domain of ␣ leads to the establishment of a small (ϳ580 Å2) ␣– interface and the formation of an ␣44 ring-like structure, which has been captured by small-angle X-ray scattering (SAXS), electron microscopy (EM), and crystallography [7, 10, 29]
Summary
Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. DATP binding to the N-terminal cone domain of ␣ leads to the establishment of a small (ϳ580 Å2) ␣– interface and the formation of an ␣44 ring-like structure, which has been captured by small-angle X-ray scattering (SAXS), electron microscopy (EM), and crystallography [7, 10, 29] In this ␣44 state, the C-terminal tail of  contacts ␣, maintaining the interaction observed in the  peptide-bound ␣2 structure [8, 29] (Fig. 1, B and C). We reason that if ring formation is responsible for dATP-induced RNR inhibition, mutations that disrupt ring stability would result in an RNR that is insensitive to dATP We test this hypothesis by employing site-directed mutagenesis to prepare RNR mutant proteins that have substitutions at the ␣– interface, including two RNR variants that were previously found to cause mutator phenotypes in vivo [31].
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