Enzymatically Active Mammalian Ribonucleotide Reductase Exists Primarily as an α6β2 Octamer

Abstract

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an ␣ 2 ␤ 2 complex consisting of R1 (␣) and R2 (␤) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/ dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (␣ 6 ␤ 2 ). The newly discovered ␣ 6 ␤ 2 complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.Ribonucleotide reductase is a key enzyme to synthesize a balanced supply of the four dNTPs used as building blocks for DNA synthesis (1). The mammalian ribonucleotide reductase reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates, which are further metabolized in the cell to become dCTP, dTTP, dATP, and dGTP. This enzyme consists of two different proteins called R1 (␣) and R2 (␤) that are both required for enzymatic activity. The R2 protein is a dimer (2 ϫ 45 kDa), and each polypeptide contains a tyrosyl radical that is generated and stabilized by an iron center. Electrons are shuttled between the tyrosyl radical and the active site in the R1 protein where the actual catalysis occurs. The R1 protein contains a substrate-binding site and two allosteric effector-binding sites termed the specificity and activity sites, respectively. ATP and dATP bind to both allosteric sites, whereas dGTP and dTTP bind to only the specificity site. In the absence of nucleotide effectors, the mammalian R1 protein is a 90-kDa monomer. Allosteric effectors that only bind to the specificity site (dTTP or dGTP) induce the formation of an enzymatically active ␣ 2 ␤ 2 complex by stimulating R1 dimer formation and R1-R2 interaction (2).The specificity site determines which substrate is to be reduced. When ATP (or dATP used at low concentration) is bound to this site, the enzyme reduces CDP and UDP. In a similar manner, dTTP stimulates GDP reduction and dGTP stimulates ADP reduction. By having this regulation, the enzyme ensures that there will be a balanced supply of all four dNTPs in the cell. The mechanism behind the specificity site function is known from experiments where the R1 dimer has been crystallized together with various allosteric effectors and substrates. When a specificity site effector is bound to one of the two R1 polypeptides, a conformational change is induced in a connecting loop that influences binding of the correct substrate to the second R1 polypeptide (3). Therefore, R1 ...

Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase

“…Allosteric regulation of this activity is key to cell survival and involves conformational changes as well as oligomeric state changes in both prokaryotic (12,14,18) and eukaryotic systems (19-23). For E. coli (12, 14, 18), mouse (20-22), yeast (19), and human (19, 23), the negative effector dATP has been linked to increases in oligomeric state with a recent gas-phase electrophoretic molecular mobility analysis (GEMMA) study estimating a molecular mass of 510 kDa for the dATP-inhibited E. coli RNR (most consistent with an α 4 β 4 state) (18), whereas for human and yeast RNR, dATP has been linked to α-hexamerization (19,22,23).To understand the role of oligomeric state in the activity regulation of this prototypic class Ia RNR from E. coli, we have combined data from four complementary structural techniques. Using small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC), we provide evidence that supports a compact α 2 β 2 structure for the active complex that can be reversibly converted via a dynamic conformational rearrangement to an inactive α 4 β 4 state in the presence of elevated dATP or protein concentrations.…”

Diversity in Overall Activity Regulation of Ribonucleotide Reductase

“…In both mechanisms, high dNTP levels mediate oligomerization into tight complexes of larger size than the common ␣ 2 ␤ 2 complex, and the ␤ 2 and ␣ 2n subunits can no longer interact in a productive way. In the eukaryotic class I enzymes, the overall activity regulation relies on two different types of ␣ 6 complexes depending on whether dATP or ATP binds to the a-site (8,9). The dATP-inhibited complex binds the ␤ 2 subunit in the center of the ␣ 6 ring in such a way that the electron transport chain between the ␤ and ␣ subunits is disrupted.…”

“…In the previous analysis of the P. aeruginosa class I RNR, the size determination of the ATP-induced oligomers was ambiguous (14), which is a common problem with gel filtration analysis of protein complexes in rapid equilibrium. In this study we have used gas-phase electrophoretic mobility macromolecule analysis (GEMMA), which is an alternative method better suited for equilibrating complexes that has successfully been used for studies of other RNRs (7,8,10).…”

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

“…The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42).…”