2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic-electron microscopy and a mechanism-based inhibitor 2′-azido-2′-deoxycytidine-5′-diphosphate (N 3 CDP) to trap a wild-type α2β2 complex of E. coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a mid-turnover state. Instead of N 3 CDP in the active site, forward RT has resulted in N 2 loss, migration of the third nitrogen from the ribose C2′ to C3′ positions, and attachment of this nitrogen to the sulfur of cysteine-225. To the best of our knowledge, this is the first time an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a pre-turnover PCET pathway and suggesting how PCET is gated at the α–β interface. This N 3 CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.
Significance Statement
Several FDA-approved cancer drugs target human ribonucleotide reductase (RNR), a radical enzyme that produces the requisite deoxyribonucleotides for DNA biosynthesis and repair. Human RNR is a class Ia enzyme that requires radical transfer (RT) from a β2 subunit to an α2 subunit on every round of turnover. Long-range RT is both a remarkable feature and an Achilles heel, given that inhibitors can intercept the radical species. Here we present a cryogenic electron microscopy (cryo-EM) structure of the best studied class Ia RNR, the enzyme from E. coli , in which α2 and β2 subunits have been trapped together using a mechanism-based inhibitor. This structure provides insight into both the mechanism of RNR inhibition and the mechanism of long-range RT.