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 alpha(2) and beta(2) subunits. The alpha subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic alpha 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.

Fig. 1.

Structure of Rnr1. (A) The dimer of apo Rnr1. Rnr1 monomers are yellow and green; dGTP (violet) and ADP (blue) from the cognate complex are shown in the specificity and catalytic sites. The three-helix insert and C-terminal insert are red and blue, respectively. (B) 1.0 σ 2F_o–F_c electron density for effector (blue density), loop 2 (red density), and substrate (green density) for the dGTP–ADP complex.

Fig. 2.

The catalytic site. (A) A schematic of ribose in the catalytic site of the dGTP–ADP complex. Carbon is yellow, oxygen red, nitrogen blue, and sulfur green. (B–E) Substrate binding (loop 2 is shown on the right). Secondary structure: dGTP–ADP, magenta; TTP–GDP, blue; AMPPNP–CDP, orange; and AMPPNP–UDP, yellow. Interacting atoms are colored as above, except phosphate, magenta; substrate carbons, cyan; protein Cα carbons, as in secondary structure. (B) Stereoview of dGTP–ADP. (C) Stereoview of TTP–GDP. (D) Stereoview of AMPPNP–CDP. (E) Stereoview of AMPPNP–UDP.

Fig. 3.

Specificity-site interactions. (A) Loop-2 rearrangements. Substrate (Left) and loop 2 and effector (Right) are shown for AMPPNP–UDP (yellow). Loop 2 is shown for apo (black), AMPPNP only (gray), and AMPPNP–CDP (orange). P294 is shown for apo and AMPPNP only. Q288 and R293 are shown for AMPPNP–CDP and AMPPNP–UDP. Cα spheres are shown for Q288, R293, and P294 in all structures. (B–E) Specificity effector binding. Colors of secondary structure cartoons are as in Fig. 2. Loop-1 carbons are yellow; Loop-2 carbons, light blue; other interacting atoms are colored as in Fig. 2, except effector carbons, which are green. (B) Stereoview of dGTP–ADP. (C) Stereoview of TTP–GDP. (D) Stereoview of AMPPNP–CDP. (E) Stereoview of AMPPNP–UDP.