title: Biophysical Studies of Bacterial Topoisomerases Substantiate Their Binding Modes to an Inhibitor.
abstract: Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between β2 and α3, and the β2 and β6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore. Electronic address: cbkang@etc.a-star.edu.sg.
authors.forename: CongBao
authors.initials: C
authors.lastname: Kang
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Joseph
authors.initials: J
authors.lastname: Cherian
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Boping
authors.initials: B
authors.lastname: Liu
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Hui Qi
authors.initials: HQ
authors.lastname: Ng
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Michelle Yueqi
authors.initials: MY
authors.lastname: Lee
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Nur Huda
authors.initials: NH
authors.lastname: Binte Ahmad
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Zhi Ying
authors.initials: ZY
authors.lastname: Poh
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Yun Xuan
authors.initials: YX
authors.lastname: Wong
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Ying Lei
authors.initials: YL
authors.lastname: Wong
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Alvin W
authors.initials: AW
authors.lastname: Hung
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hill
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A(∗)STAR), Singapore. Electronic address: thkeller@etc.a-star.edu.sg.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Keller
mesh_terms: D000595:Amino Acid Sequence; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa; D012996:Solutions; D013296:Streptococcus pneumoniae; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D012996:Solutions; D027101:DNA Topoisomerase IV