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Its N-1-pyridinyl-, C-7-azetidinyl- and C-8-chlorine substituents confer enhanced antibiotic activity against bacteria resistant to other fluoroquinolones, but its mode of action is unclear. Here we present the X-ray crystal structures of a delafloxacin-DNA cleavage complex obtained by co-crystallization with <jats:italic>Streptococcus pneumoniae<\/jats:italic> topo IV using a graphene nucleant and solved at 2.0 and 2.4\u2009\u00c5 resolution. The two Mg<jats:sup>2+<\/jats:sup>-chelated delafloxacin molecules intercalated at the DNA cleavage site are bound in an unusual conformation involving interacting out-of-plane N-1-aromatic- and C-8-chlorine- substituents. The unprecedented resolution allows comprehensive imaging of water-metal ion links integrating enzyme and DNA through drug-bound and active-site Mg<jats:sup>2+<\/jats:sup> ions plus the discovery of enzyme-bound K<jats:sup>+<\/jats:sup> ions. Our studies on delafloxacin action suggest that intrinsic target affinity contributes to its activity against quinolone-resistant bacteria.<\/jats:p>","DOI":"10.1038\/s41467-025-60688-3","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T12:33:23Z","timestamp":1751373203000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Structural basis of topoisomerase targeting by delafloxacin"],"prefix":"10.1038","volume":"16","author":[{"given":"Shabir","family":"Najmudin","sequence":"first","affiliation":[]},{"given":"Xiao-Su","family":"Pan","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0009-0005-8674-5170","authenticated-orcid":false,"given":"Beijia","family":"Wang","sequence":"additional","affiliation":[]},{"given":"Lata","family":"Govada","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2861-2458","authenticated-orcid":false,"given":"Naomi E.","family":"Chayen","sequence":"additional","affiliation":[]},{"given":"Noelia","family":"Rubio","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9384-9043","authenticated-orcid":false,"given":"Milo S. 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