Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome
Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome
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DOI：
10.1016/j.abb.2017.10.020
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发表时间：
2017-12-15
影响因子：
3.9
通讯作者：
Pey, Angel Luis
中科院分区：
文献类型：
--
作者：
Claveria-Gimeno, Rafael;Velazquez-Campoy, Adrian;Pey, Angel Luis
人类黄素蛋白的稳定性很大程度上取决于黄素水平，尽管人们对这种关系的结构和能量基础知之甚少。在这里，我们报告了对 FAD 与这种关系最具代表性的例子之一 NAD(P)H:醌氧化还原酶 1 (NQO1) 结合的热力学的深入分析。 NQO1 是一种与 FAD 紧密结合的二聚酶，结合后会引发较大的结构变化。常见的癌症相关多态性 (P187S) 严重损害 FAD 结合。我们表明，当应用于不同组的量热分析和 NQO1 变体时，FAD 结合可以通过明确结合结合协同性的热力学模型得到很好的描述，从而深入了解癌症相关 P187S 的体外和细胞内的影响、其抑制突变 H8OR 以及 NQO1 C 端结构域在调节结合协同性和能量方面的作用。此外，我们发现 FAD 与 NQO1 的结合对生理相关的环境条件非常敏感，例如磷酸盐缓冲液和盐的存在。总体而言，我们的结果有助于在分子水平上理解 NQO1 稳定性与细胞内 FAD 水平波动之间的联系，并支持 FAD 结合能量和协同性与 apo-NQO1 构象整体的动态性质从根本上相关的观点。
The stability of human flavoproteins strongly depends on flavin levels, although the structural and energetic basis of this relationship is poorly understood. Here, we report an in-depth analysis on the thermodynamics of FAD binding to one of the most representative examples of such relationship, NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a dimeric enzyme that tightly binds FAD, which triggers large structural changes upon binding. A common cancer-associated polymorphism (P187S) severely compromises FAD binding. We show that FAD binding is described well by a thermodynamic model explicitly incorporating binding cooperativity when applied to different sets of calorimetric analyses and NQO1 variants, thus providing insight on the effects in vitro and in cells of cancer-associated P187S, its suppressor mutation H8OR and the role of NQO1 C-terminal domain to modulate binding cooperativity and energetics. Furthermore, we show that FAD binding to NQO1 is very sensitive to physiologically relevant environmental conditions, such as the presence of phosphate buffer and salts. Overall, our results contribute to understanding at the molecular level the link between NQO1 stability and fluctuations of FAD levels intracellularly, and supports the notion that FAD binding energetics and cooperativity are fundamentally linked with the dynamic nature of apo-NQO1 conformational ensemble.