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 alpha(2) and beta(2) subunits. Rnr1, the alpha subunit, contains regulatory and catalytic sites; Rnr2, the beta 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.

Fig. 1.

Rnr1 complexes. (A) The dimer of Rnr1 is displayed as a cartoon with the monomers colored blue and green. Surfaces for Rnr2pep and Rnr4pep are displayed in yellow and pink, respectively, and AMPPNP (gray) and GemdP (red) are shown binding to the specificity and catalytic sites. (B and C) The 2F_o–F_c electron density contoured at 1.0 σ for CDP (B) and GemdP (C).

Fig. 2.

Catalytic-site interactions. (A) Stereoview of CDP (orange). Interacting atoms: oxygen, red; nitrogen, blue; phosphate, magenta; sulfur, green; substrate carbons, cyan; protein non-Cα carbons, yellow; Cα carbons, as secondary structure, orange. (B) Stereoview of GemdP. Interacting atoms are colored as in A, except that sulfur is orange; Cα carbons, as secondary structure, are yellow; and fluorines are gray. (C) Ligand plot of CDP ribose interactions. Colors are as in A, except that carbons are yellow. (D) Ligand plot of GemdP interactions. The van der Waals contact to L427 is omitted for clarity. (E) Stereoview of loop-2 superposition of AMPPNP–CDP (orange) and AMPPNP–GemdP (yellow). Substrate/inhibitor is seen on the left, and the effector is on the right. The color scheme is the same as in C, but fluorine is black.

Fig. 3.

Binding of Rnr2 and Rnr4 peptides to Rnr1. The structures of Rnr1–Rnr2pep complex in stereo (A), Rnr1–Rnr4pep complex in stereo (B), and Rnr4pep superimposed on the Rnr2pep complex (C). Cα backbones are shown for Rnr2pep (yellow) and Rnr4pep (pink), and nearby helices are drawn from Rnr1 from the Rnr2pep complex (green). (D) E. coli Rnr2pep (gray) superimposed on yeast Rnr2p (yellow) and Rnr1 (green). (E) Sequence alignment through Rnr2pep-binding site. Secondary structure is shown for E. coli (above) and yeast (below). Residues that interact with the peptide are colored. The Rnr2pep residues binding a given subsite are listed below the corresponding sequence. (F) Mouse Rnr2pep (cyan) superimposed on yeast Rnr2pep (yellow); some side chains are omitted for clarity.