Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase

Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATP and dATP bind at an additional allosteric site 40 A away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned >300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization.