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

Area of Science:

• Biochemistry
• Structural Biology
• Cancer Therapeutics

Background:

• Ribonucleotide reductase (RNR) is essential for DNA synthesis and rapidly proliferating cells, making it a key target in cancer therapy.
• Eukaryotic RNR is a heterotetramer composed of alpha(2) and beta(2) subunits, with Rnr1 (alpha) containing catalytic and regulatory sites and Rnr2 (beta) harboring the essential diferric-tyrosyl radical.

Purpose of the Study:

• To elucidate the structural mechanisms of eukaryotic Rnr1 interaction with gemcitabine diphosphate (GemdP) and its own subunits (Rnr2 and Rnr4).
• To provide a structural basis for understanding RNR function and developing novel RNR-targeted cancer therapies.

Main Methods:

• X-ray crystallography was employed to determine the three-dimensional structures of Saccharomyces cerevisiae Rnr1.
• Structures were obtained for Rnr1 in complex with GemdP, an Rnr2-derived peptide, and an Rnr4-derived peptide.

Main Results:

• GemdP exhibits distinct binding interactions compared to cytidine diphosphate, driven by its geminal fluorines, leading to significant shifts in the ribose and base, and altered conformations of the substrate-specificity loop.
• Rnr2 and Rnr4 peptides bind to Rnr1 via unique modes not observed in prokaryotic RNR, inhibiting enzyme assembly.
• The Rnr2 peptide adopts a conformation consistent with previous NMR studies and binds to conserved residues, suggesting conserved Rnr1-Rnr2 interaction mechanisms across eukaryotes.

Conclusions:

• The unique binding of GemdP to Rnr1 provides a structural foundation for understanding gemcitabine's mechanism of action and resistance.
• The distinct binding modes of Rnr2 and Rnr4 peptides offer insights into RNR subunit assembly regulation.
• These structural findings pave the way for structure-based drug design targeting RNR for improved cancer therapeutics.