Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant
Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant
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DOI：
10.1021/acs.biochem.9b00653
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发表时间：
2019-11-12
期刊：
影响因子：
2.9
通讯作者：
Fierke, Carol A.
中科院分区：
文献类型：
--
作者：
Leng, Katherine R. Welker;Castaneda, Carol Ann;Fierke, Carol A.
组蛋白去乙酰化酶（HDAC）酶催化去除乙酰赖氨酸翻译后修饰，经常被翻译后修饰。HDAC8在去乙酰化酶结构域的保守残基丝氨酸39处被磷酸化，导致催化活性降低。HDAC8在S39的磷酸化在其位置和功能上是独特的，可能代表了一种新的去乙酰化调节模式。为了更好地了解HDAC8磷酸化对酶结构和功能的影响，我们对S39E HDAC8拟磷突变体进行了晶体学、动力学和分子动力学研究。这种突变降低了源自乙酰化核蛋白和细胞质蛋白的肽的去乙酰化水平。然而，影响的大小取决于肽序列和活性位点金属离子[Zn(II) vs Fe(II)1的身份，突变体的k(cat)/ k - m值比野生型HDAC8降低了9- 200倍。此外，活性位点金属离子的解离速率常数提高了约10倍。S39E HDAC8与抑制剂Droxinostat配合结晶，揭示了S39的磷酸化，如谷氨酸侧链所模拟的那样，通过L1环的扭曲扰乱了局部结构。对S39E和磷酸化S39 HDAC8的分子动力学模拟表明，L1环的扰动可能是由于D29和S39之间失去了氢键。此外，S39的扰动引起结构变化，通过蛋白质支架传播，影响活性部位的功能。这些数据表明，磷酸化通过影响配体结合、催化效率和底物选择性，对HDAC8起着重要的调节作用。
Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)1, with the value of k(cat)/K-M for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by similar to 10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.