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

Michalczyk, Elizabeth; Pakosz-Stępień, Zuzanna; Liston, Jonathon D.; Gittins, Olivia; Pabis, Marta; Heddle, Jonathan G.; Ghilarov, Dmitry

Abstract

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 β-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.

Citation

Michalczyk, E., Pakosz-Stępień, Z., Liston, J. D., Gittins, O., Pabis, M., Heddle, J. G., & Ghilarov, D. (2024). Structural basis of chiral wrap and T-segment capture by Escherichia coli DNA gyrase. Proceedings of the National Academy of Sciences, 121(49), Article e2407398121. https://doi.org/10.1073/pnas.2407398121

• Journal Article Type: Article
• Acceptance Date: Oct 17, 2024
• Online Publication Date: Nov 26, 2024
• Publication Date: Dec 3, 2024
• Deposit Date: Nov 27, 2024
• Publicly Available Date: Nov 27, 2024
• Journal: Proceedings of the National Academy of Sciences
• Print ISSN: 0027-8424
• Electronic ISSN: 1091-6490
• Publisher: National Academy of Sciences
• Peer Reviewed: Peer Reviewed
• Volume: 121
• Issue: 49
• Article Number: e2407398121
• DOI: https://doi.org/10.1073/pnas.2407398121
• Keywords: Models, Molecular, Escherichia coli - metabolism - enzymology - genetics, DNA Gyrase - metabolism - chemistry, Moxifloxacin - chemistry, Cryoelectron Microscopy, Catalytic Domain, Topoisomerase II Inhibitors - chemistry - pharmacology, antibiotics, DNA crossover, topoisomerase, DNA, Superhelical - metabolism - chemistry, molecular machine, Nucleic Acid Conformation, DNA-binding protein, DNA, Bacterial - metabolism, Fluoroquinolones - chemistry - pharmacology
• Public URL: https://durham-repository.worktribe.com/output/3107520