Abstract:
The N-terminal region of the human lysine-specific demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple posttranslational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1\'s own electropositive NLS amino acids 107 to 120 inhibit demethylation activity on a model histone 3 lysine 4 dimethyl (H3K4me2) peptide (Kiapp ∼ 3.3 μM) and histone 3 lysine 4 dimethyl nucleosome substrates (IC50 ∼ 30.4 μM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, intrinsically disordered regions, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.
摘要:
人赖氨酸特异性去甲基酶1(LSD1)的N端区域没有预测的结构元件,包含一个核定位信号(NLS),经历多个翻译后修饰(PTM),并充当蛋白质-蛋白质相互作用的枢纽。该固有无序区域(IDR)从核心LSD1结构延伸,位于催化活性位点的顶部,并且已知对于催化是可有可无的。这里,我们显示了全长和N端缺失的LSD1之间的差异核小体结合,并确定N端的保守的NLS和PTM含有元件含有α螺旋结构,这种保守的元素会影响去甲基化。酶分析显示,LSD1自身的阳性NLS氨基酸107-120抑制模型组蛋白3赖氨酸4二甲基(H3K4me2)肽(Kiapp〜3.3μM)和H3K4me2核小体底物(IC50〜30.4μM)的去甲基化活性,可能模仿组蛋白H3尾巴。Further,当相同的,抑制性NLS区含有拟磷酸修饰,抑制部分缓解。根据这些结果和生物物理数据,提出了LSD1催化的去甲基化反应的调节机制,其中NLS介导的自抑制可以通过静电相互作用发生,并通过NLS附近发生的磷酸化部分缓解。一起来看,结果突出了PTM的动态和协同作用,IDR,和LSD1活性位点附近的结构化区域,并引入了磷酸化介导的NLS区域可以起到微调染色质修饰酶活性的作用。