Mass Spectrometry Reveals That the Antibiotic Simocyclinone D8 Binds to DNA Gyrase in a "Bent-Over" Conformation: Evidence of Positive Cooperativity in Binding
Mass Spectrometry Reveals That the Antibiotic Simocyclinone D8 Binds to DNA Gyrase in a "Bent-Over" Conformation: Evidence of Positive Cooperativity in Binding
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DOI：
10.1021/bi101691k
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发表时间：
2011-05-03
期刊：
影响因子：
2.9
通讯作者：
McKay, Adam R.
中科院分区：
文献类型：
--
作者：
Edwards, Marcus J.;Williams, Mark A.;McKay, Adam R.
DNA拓扑异构酶是控制DNA拓扑结构的酶，并且是抗微生物和抗癌药物的重要靶标。在这里，我们提出了一个质谱研究的拓扑异构酶DNA促旋酶的A亚基和双功能抑制剂Simocyclinone D8（SD8），从链霉菌分离的抗生素之间形成的复合物。这些研究表明，在一种替代的相互作用模式，发现由X射线晶体学，每个亚基结合一个单一的双功能抑制剂与单独的结合口袋的两端SD8。促旋酶亚基形成组成性二聚体，并且两个双功能配体与二聚体的结合中存在强的别构协同性。我们表明，通过紧结合方法的扩展，质谱数据可以拟合到合作结合的一般模型，从而严格确定解离常数和协同度。这种一般的方法将适用于其他系统与多个结合位点，并突出了质谱作为一个强大的新兴工具，解开生物分子相互作用的复杂性的作用。
DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the tight-binding approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometrys role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.