Crystallization and structure determination of topoisomerase IV protein-DNA-drug complexes with the aid of novel nucleants
File(s)
Author(s)
Wang, Beijia
Type
Thesis
Abstract
Streptococcus pneumoniae is a major bacterial pathogen responsible for numerous life-threatening conditions. The emergence of antimicrobial resistance has threatened the effectiveness of existing treatments, highlighting the importance of understanding mechanisms underlying resistance and to develop novel antibacterials. Bacterial type II topoisomerases, gyrase and topoisomerase IV (topo IV), are essential enzymes for the regulation of DNA topology during DNA replication, transcription and chromosome segregation. These enzymes are targets of an important class of clinically relevant antibacterials, fluoroquinolones. Fluoroquinolones act by stabilising the topoisomerase complex in the DNA-cleaved state, preventing the DNA from resealing and ultimately resulting in bacterial cell death. In this thesis, the first X-ray structures were obtained from topo IV-DNA cleavage complexes with delafloxacin and with gemifloxacin, which are new-approved fluoroquinolones. These structures revealed the multiple interactions mediated by these fluoroquinolones involving the enzyme, DNA, magnesium (Mg2+) ions and water molecules. Delafloxacin and gemifloxacin exhibited different binding interactions compared to other fluoroquinolones, which could result in tighter binding and hence explain the potency of these fluoroquinolones against resistant bacteria. Furthermore, a topo IV-DNA-delafloxacin complex structure was solved to 2.0 Å with the aid of a graphene-based nucleant, achieving the highest resolution to date for any fluoroquinolone complexes. At this high resolution, clear locations of water molecules coordinated the drug-bound Mg2+ ions were revealed, suggesting the involvement of bridging water molecules in fluoroquinolone action. The identity and location of the Mg2+ ions were confirmed experimentally using long-wavelength X-ray crystallography studies. These experiments also led to the discovery of two pairs of potassium ions and one pair of chloride ions with possible roles in the structure and function of topoisomerases. Overall, this work has provided new structural insights into how fluoroquinolones target topoisomerases, which could help in the design of future topoisomerase-targeting therapeutics, an essential approach in combating antimicrobial resistance.
Version
Open Access
Date Issued
2025-04-11
Date Awarded
2026-02-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Chayen, Naomi
Sanderson, Mark
Neidle, Stephen
Publisher Department
Department of Metabolism, Digestion and Reproduction
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)