2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP

• 成果类型: Article
• 署名作者: Westmoreland, Dana E.; Feliciano, Patricia R.; Kang, Gyunghoon; Cui, Chang; Kim, Albert; Stubbe, Joanne; Nocera, Daniel G.; Drennan, Catherine L.
• 署名单位: Massachusetts Institute of Technology (MIT); Howard Hughes Medical Institute; Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); Harvard University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12535
• DOI: 10.1073/pnas.2417157121
• 发表日期: 2024-11-05
• 关键词: coupled electron-transfer; escherichia-coli; radical transfer; diphosphate reductase; subunit interface; crystal-structure; active-site; inactivation; protein; intermediate

摘要

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (beta 2) from the subunit housing the active site (alpha 2), requiring the formation of a short-lived alpha 2 beta 2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (similar to 32-angstrom) 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 (N3CDP) to trap a wild-type alpha 2 beta 2 complex of Escherichia coli class Ia RNR. We find that one alpha subunit has turned over and that the other is trapped, bound to beta in a midturnover state. Instead of N3CDP 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. In this study, 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 preturnover PCET pathway and suggesting how PCET is gated at the alpha-beta interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 angstrom) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.