Structures of eukaryotic ribonucleotide reductase I define gemcitabine diphosphate binding and subunit assembly

Abstract

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of ␣2 and ␤2 subunits. Rnr1, the ␣ subunit, contains regulatory and catalytic sites; Rnr2, the ␤ subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from Saccharomyces cerevisiae: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1-Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.allosteric regulation ͉ crystallography ͉ dNTP ͉ chemotherapy ͉ gemcitabine R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, essential precursors of DNA synthesis. Crucial for rapidly proliferating cells, RNR is a target for anticancer (1, 2) and antiviral (2, 3) drugs. Gemcitabine, an analogue of deoxycytidine (2Ј-2Ј-difluorodeoxycytidine), is sequentially phosphorylated to the 5Ј-monophosphate form by deoxycytidine kinase and to difluorodeoxycytidine 5Ј-diphosphate (GemdP) by uridylate-cytidylate monophosphate kinase. In the presence of reductants, GemdP inactivates Rnr1. In the absence of reductants, with prereduced Rnr1 and Rnr2, inhibition occurs from the loss of the tyrosyl radical in Rnr2 (1). Recently, GemdP has been shown to inactivate both human R1 and R2 (JoAnne Stubbe, personal communication). Inhibition of RNR by GemdP leads to reduction of the pool of deoxyribonucleotide 5Ј-diphosphates available for DNA synthesis, presumably favoring incorporation of the gemcitabine triphosphate metabolite by DNA polymerase ␣, preventing chain elongation (4, 5).RNRs require unusual metallocofactors to initiate radicalbased nucleotide reduction and are divided into three classes based on their cofactor. Class I RNR, found in all eukaryotes, is a heterooligomer of ␣ 2 and ␤ 2 subunits (6). In eukaryotes, the ␣ subunit, called Rnr1, c...

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

“…Additionally, structure-activity relationship studies focused on the naphthalene and benzene ring systems will provide insight into variations that could improve the compound's potency toward hRR. Finally, growing the compound with fragments reaching toward the loop 2 region of the C-site will improve additional binding interactions with hRRM1 and potentially lead to improved inhibitory potency (9,33,34,40) (Fig. S4).…”

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

“…In all known crystals structures of R1, the C-terminal regions are thermally labile and structurally undefined (18,19,29,30). Thus, the location(s) of the C terminus in the tertiary structure of R1 is not known.…”

Regulators of Cellular Levels of Histone Acetylation in Saccharomyces cerevisiae

“…The VPS genes are involved in a catabolic membrane-trafficking phenomenon termed autophagy that occurs in response to adverse environmental signals such as nutrient or hormonal deprivation and is linked to the carcinogenic potential (Abeliovich and Klionsky 2001). Finally, activity of ribonucleotide reductase including its large subunit, Rnr1 (identified in our screen), is crucial for rapidly dividing cells and is a target for anticancer and antiviral therapies (Xu et al 2006). Taken together with our data, it is conceivable that the effect of these genes on cancer may be mediated partly through regulation of histone acetylation.…”