Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation.
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.
Xu
Hai
H
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, M407 Walters Life Sciences, Knoxville, TN 37996-0840, USA.
Faber
Catherine
C
Uchiki
Tomoaki
T
Fairman
James W
JW
Racca
Joseph
J
Dealwis
Chris
C
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003854:Deoxyribonucleotides / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology
D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases