Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
J Biol Chem. 2011 Feb 11;286(6):4392-403. doi: 10.1074/jbc.M110.193466. Epub 2010 Dec 1.
Rev-erbβ is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle, and proinflammatory responses. Here, we demonstrate that a thiol-disulfide redox switch controls the interaction between heme and the ligand-binding domain of Rev-erbβ. The reduced dithiol state of Rev-erbβ binds heme 5-fold more tightly than the oxidized disulfide state. By means of site-directed mutagenesis and by UV-visible and EPR spectroscopy, we also show that the ferric heme of reduced (dithiol) Rev-erbβ can undergo a redox-triggered switch from imidazole/thiol ligation (via His-568 and Cys-384, based on a prior crystal structure) to His/neutral residue ligation upon oxidation to the disulfide form. On the other hand, we find that change in the redox state of iron has no effect on heme binding to the ligand-binding domain of the protein. The low dissociation constant for the complex between Fe(3+)- or Fe(2+)-heme and the reduced dithiol state of the protein (K(d) = ∼ 20 nM) is in the range of the intracellular free heme concentration. We also determined that the Fe(2+)-heme bound to the ligand-binding domain of Rev-erbβ has high affinity for CO (K(d) = 60 nM), which replaces one of the internal ligands when bound. We suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance. Heme dissociation from Rev-erbβ has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions. We propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbβ and altering its transcriptional activity.
REV-ERBβ 是一种血红素结合的核激素受体,可抑制广泛参与代谢、昼夜节律和促炎反应的靶基因。在这里,我们证明了一种硫醇-二硫键氧化还原开关控制着血红素与 REV-ERBβ 的配体结合域之间的相互作用。REV-ERBβ 的还原二硫状态比氧化的二硫状态更紧密地结合血红素 5 倍。通过定点突变和紫外可见光谱和 EPR 光谱,我们还表明,还原(二硫)REV-ERBβ 的高铁血红素可以通过氧化还原触发开关从咪唑/硫醇键合(通过先前的晶体结构中的 His-568 和 Cys-384)转变为 His/中性残基键合,形成二硫形式。另一方面,我们发现铁的氧化还原状态变化对血红素与蛋白质的配体结合域的结合没有影响。Fe(3+)-或 Fe(2+)-血红素与蛋白质的还原二硫状态之间复合物的低解离常数(Kd = ∼20 nM)在细胞内游离血红素浓度范围内。我们还确定,与 REV-ERBβ 的配体结合域结合的 Fe(2+)-血红素有高亲和力与 CO(Kd = 60 nM)结合,当结合时取代其中一个内部配体。我们认为,这种硫醇-二硫键氧化还原开关是氧化应激与昼夜节律和/或代谢失衡相关的一种机制。血红素从 REV-ERBβ 的解离已被证明可以在细胞内氧化还原条件发生变化时,解除对靶基因的表达抑制。我们提出,氧化应激导致半胱氨酸(s)氧化,从而从 REV-ERBβ 释放血红素并改变其转录活性。