Heme binding to human CLOCK affects interactions with the E-box
Heme binding to human CLOCK affects interactions with the E-box
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DOI：
10.1073/pnas.1905216116
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发表时间：
2019-10-01
影响因子：
11.1
通讯作者：
Raven, Emma L.
中科院分区：
文献类型：
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作者：
Freeman, Samuel L.;Kwon, Hanna;Raven, Emma L.
生物钟是一种内源性的时间保持系统，在动物和植物以及一些细菌中普遍存在。在哺乳动物中，生物钟通过2个基本的螺旋-环-螺旋PER-ARNT-SIM（bHLH-PAS）结构域蛋白-CLOCK和BMAL 1调节睡眠-觉醒周期。有新的证据表明，血红素影响昼夜节律控制，通过血红素结合到各种昼夜节律蛋白，但调节机制在很大程度上是未知的。在这项工作中，我们研究血红素与人类时钟（hCLOCK）的相互作用。我们给出了hCLOCK的PAS-A结构域的晶体结构，并检查了血红素与PAS-A和PAS-B结构域的结合。紫外-可见和电子顺磁共振光谱是一致的，在溶液中的氧化（铁）PAS-A蛋白质的双组氨酸连接血红素物种，并通过诱变，我们确定His 144作为配体的血红素。有证据表明血红素口袋中的灵活性，这可能会在20 K时产生额外的Cys轴向配体（His/Cys配位）。使用DNA结合试验，我们证明，血红素破坏结合的时钟，其E盒DNA目标。证据是一个构象移动的蛋白质框架，这是连接到血红素连接的变化，并有能力影响结合的E-盒。在hCLOCK结构框架内，这将提供血红素依赖性转录调节的机制。
The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.