Structural basis of chiral wrap and T- segment capture by Escherichia coli DNA gyrase

• 成果类型: Article
• 署名作者: Michalczyk, Elizabeth; Pakosz-Stepen, Zuzanna; Liston, Jonathon D.; Gittins, Olivia; Pabis, Marta; G. Heddle, Jonathan; Ghilarov, Dmitry
• 署名单位: Jagiellonian University; Jagiellonian University; Durham University; UK Research & Innovation (UKRI); Biotechnology and Biological Sciences Research Council (BBSRC); John Innes Center
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14606
• DOI: 10.1073/pnas.2407398121
• 发表日期: 2024-12-03
• 关键词: resistance-determining region; n-terminal fragment; cryo-em structure; topoisomerase-ii; atp-binding; strand passage; nucleotide-binding; crystal-structure; mechanism; cleavage

摘要

Type II topoisomerase DNA gyrase transduces the energy of ATP hydrolysis into the negative supercoiling of DNA. The postulated catalytic mechanism involves stabilization of a chiral DNA loop followed by the passage of the T- segment through the temporarily cleaved G- segment resulting in sign inversion. The molecular basis for this is poorly understood as the chiral loop has never been directly observed. We have obtained high- resolution cryoEM structures of Escherichia coli gyrase with chirally wrapped 217 bp DNA with and without the fluoroquinolone moxifloxacin (MFX). Each structure constrains a positively supercoiled figure- of- eight DNA loop stabilized by a GyrA beta- pinwheel domain which has the structure of a flat disc. By comparing the catalytic site of the native drug- free and MFX-bound gyrase structures both of which contain a single metal ion, we demonstrate that the enzyme is observed in a native precatalytic state. Our data imply that T- segment trapping is not dependent on the dimerization of the ATPase domains which appears to only be possible after strand passage has taken place.