Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation

Abstract

Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of ␣2 and ␤2 subunits. The ␣ subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic ␣ subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP-CDP-, AMPPNP-UDP-, dGTP-ADP-and TTP-GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue-substrate interactions differ substantially between yeast and T. maritima. In most effector-substrate complexes, water molecules help mediate substrate-loop 2 interactions. Finally, the substrate ribose binds with its 3 hydroxyl closer than its 2 hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2 hydroxyl.ribonucleotide reductase ͉ allosteric regulation ͉ crystallography ͉ DNA synthesis ͉ dNTP pools E ukaryotic ribonucleotide reductase (RNR) is an enzyme composed of ␣ 2 and ␤ 2 subunits that catalyzes a crucial step of de novo DNA synthesis by converting nucleoside diphosphates to deoxynucleoside diphosphates (1, 2). Tight control of dNTP pools is vital for cell viability; because of the crucial role RNR plays in balancing the relative levels of dNTPs, it is highly regulated transcriptionally (3), allosterically (4-6), by compartmentalization of the various subunits within the cell (7,8), and, in Saccharomyces cerevisiae, by its protein inhibitor Sml1 (9-12). The molecular basis for these processes is not fully understood.Rnr1, the ␣ subunit of RNR, contains the catalytic site, the substrate-specificity site, and the activity site (Fig. 1A), whereas the ␤ subunit houses a tyrosyl radical required for RNR activity (13,14). An elegant mechanism of specificity cross-talk determines substrate preference based on the nucleotide effector bound at the specificity site (4, 15, 16). Brown and Reichard (2) have proposed that the effectors ATP and dATP bind at the activity site and activate or inhibit, respectively. They also bind at the specificity site and select for pyrimidine substrates, whereas thymin triphosphate (TTP) and dGT...

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule

“…This is based on previous studies where Arg-293 and Gln-288 (hRR numbering), or their equivalent residues in S. cerevisiae, E. coli, and Thermotoga maritima RRs, play an important role in substrate recognition (32)(33)(34)41). In particular, in T. maritima and E. coli RR1 structures, arginine forms a salt bridge with the β-phosphate of the substrate, which is a crucial interaction.…”

“…The naphthalene ring is buried within a pocket containing a mixture of hydrophobic and hydrophilic residues. Usually, when nucleoside diphosphate substrates are bound, this pocket will form multiple hydrogen bonds with the phosphate groups, primarily through the backbone atoms of Ser-202, Ser-606, and Ser-610 (9,32). The naphthalene ring interacts with four hydrophobic residues: Met-602, Ala-201, Ala-605, and Leu-428; and three hydrophilic residues: Cys-218, Ser-606, and Thr-607 (Table S3).…”

Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1

“…The inhibition of R1 by Sml1 depends on Sml1-R1 association because mutations in SML1 disrupting its R1-binding ability abolish the inhibition (17). Crystallographic studies of the R1s from E. coli and S. cerevisiae reveal three domains in the protein: the N-terminal helical domain, the 10-stranded ␣/␤-barrel domain, and the C-terminal domain of less-defined structure (18,19). The active site is located in the center of the protein between the N-terminal and the barrel domains, wherein a redox-active cysteine pair (Cys-225/Cys-462 of the E. coli R1 and Cys-218/ Cys-443 of the yeast R1) converts from a free dithiol form in the reduced R1 (active form) to a disulfide-bonded form in the oxidized R1 (inactive form) after each reduction cycle (20).…”

Nonself Recognition Through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

“…Removal of six residues from the C terminus end [hygunPA (788-917)], including the putative redox-active and catalytically required C terminus cysteine pair, resulted in a loss of incompatibility activity. These residues lie in the putative flexible C terminus arm of R1 as defined in yeast (Xu et al 2006;Zhang et al 2007).…”

“…The tertiary structure of the large RNR subunit from Saccharomyces cerevisiae (Rnr1p) is similar to the E. coli ortholog (Xu et al 2006), while the predicted protein sequence of in N. crassa is 85% similar to Rnr1p. A major departure from the similarity of these proteins occurs in the C terminus (Smith et al 2000b).…”