Department of Physical Chemistry, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain.
Int J Biol Macromol. 2019 Apr 1;126:1223-1233. doi: 10.1016/j.ijbiomac.2019.01.016. Epub 2019 Jan 4.
Ion binding to biomacromolecules can modulate their activity and stability in vivo. It is of particular interest to understand the structural and energetic basis of anion binding to functional sites of biomacromolecules. In this work, binding of anions to the FAD binding pocket of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with cancer due to a common polymorphism causing a P187S amino acid substitution, was investigated. It is known that NQO1 stability in vivo is strongly modulated by binding of its flavin cofactor. Herein, binding and protein stability analyses were carried out to show that anion binding to the apo-state of NQO1 P187S inhibits FAD binding with increasing strength following the chaotropic behavior of anions. These inhibitory effects were significant for some anions even at low millimolar concentrations. Additional pH dependent analyses suggested that protonation of histidine residues in the FAD binding pocket was not critical for anion or flavin binding. Overall, this detailed biophysical analysis helps to understanding how anions modulate NQO1 functionality in vitro, thus allowing hypothesize that NQO1 stability in vivo could be modulated by differential anion binding and subsequent inhibition of FAD binding.
离子与生物大分子的结合可以调节它们在体内的活性和稳定性。了解阴离子与生物大分子功能部位结合的结构和能量基础特别有趣。在这项工作中，研究了阴离子与人 NAD(P)H：醌氧化还原酶 1（NQO1）的 FAD 结合口袋的结合，NQO1 是一种由于常见的导致 P187S 氨基酸取代的多态性而与癌症相关的黄素蛋白。众所周知，NQO1 的黄素辅因子的结合强烈调节其在体内的稳定性。在此，进行了结合和蛋白质稳定性分析，以表明阴离子与 NQO1 P187S 的apo 状态结合，随着阴离子的离液行为，以越来越强的强度抑制 FAD 结合。即使在低毫摩尔浓度下，这些抑制作用对某些阴离子也非常显著。额外的 pH 依赖性分析表明，FAD 结合口袋中组氨酸残基的质子化对于阴离子或黄素结合不是关键的。总的来说，这种详细的生物物理分析有助于理解阴离子如何在体外调节 NQO1 的功能，从而可以假设体内 NQO1 的稳定性可以通过不同的阴离子结合和随后对 FAD 结合的抑制来调节。
