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首页> 外文期刊>Biochemistry >Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant
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Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant

机译：组蛋白脱乙酰酶8的磷酸化：磷染色素S39E突变体的结构和机械分析

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摘要

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.
机译：催化除去乙酰赖氨酸后翻译后修饰的组蛋白脱乙酰化酶（HDAC）酶经常翻译翻译后修饰。 HDAC8在保守的残余物丝氨酸39处在脱乙酰酶结构域内磷酸化，这导致催化活性降低。 S39的HDAC8磷酸化在其位置和功能中是独特的，并且可以代表一种新的脱乙酰化调节模式。为了更好地了解HDAC8磷酸化对酶结构和功能的影响，我们进行了S39E HDAC8磷光瘤突变体的晶体，动力学和分子动力学研究。该突变降低了衍生自乙酰化核和细胞质蛋白的肽的脱乙酰化水平。然而，效果的大小取决于肽序列和活性位点金属离子的同一性[Zn（II）Vs Fe（II）1，具有K（猫）/ km的突变体递减9-至与野生型HDAC8相比，200倍。此外，活性位点金属离子的解离速率常数随20倍而增加。 S39E HDAC8与抑制剂Droxinostat结晶，揭示了S39的磷酸化，由谷氨酸侧链模仿，通过L1环的变形磨削局部结构。 S39E和磷酸化S39 HDAC的分子动力学模拟表明，由于D29和S39之间的氢键丧失，可能发生L1环的扰动。此外，S39扰动会导致通过蛋白质支架繁殖以影响活性位点的功能的结构变化。这些数据表明，通过影响配体结合，催化效率和底物选择性，磷酸化对HDAC8发挥着重要的调节作用。

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