Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
Postgraduate School of Molecular Medicine, Warsaw, Poland.
Nucleic Acids Res. 2021 Feb 22;49(3):1581-1596. doi: 10.1093/nar/gkaa1266.
DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.
DNA 拓扑异构酶 II 是一种主要存在于细菌中的酶，是多种“毒物”的靶标，包括天然产物毒素（如 albicidin、microcin B17）和临床上重要的合成分子（如氟喹诺酮类）。对这两类药物的耐药性都可以由五肽重复蛋白（PRP）介导。尽管进行了长期研究，但这些保护性 PRP 的作用机制尚不清楚。我们表明，一种 PRP，QnrB1 提供了针对氟喹诺酮类药物的特异性保护，而这严格依赖于拓扑异构酶 II 的 ATP 水解。QnrB1 与 GyrB 蛋白结合，并刺激分离的 GyrB N 端 ATP 酶结构域（GyrB43）的 ATP 酶活性。我们使用光反应氨基酸的定点掺入来探测 QnrB1 的结合位点，并将交联映射到 GyrB43 蛋白上。我们提出了一个模型，其中 QnrB1 的结合变构促进了氟喹诺酮类药物从切割复合物中的解离。
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