MOE Key Laboratory of Biosystems Homeostasis & Protection, College of Life Sciences, Zhejiang University, Hangzhou 310000, China.
Protein Pept Lett. 2022;29(10):891-899. doi: 10.2174/0929866529666220819121911.
Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU.
We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans.
LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses.
The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress.
Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.
组蛋白样蛋白是广泛存在于原核生物中的小分子 DNA 结合蛋白。这些蛋白质在细胞结构和过程中具有多种功能，包括核基质的形态稳定性、DNA 紧凑性、DNA 复制和 DNA 修复。耐辐射球菌是一种极端微生物，具有非凡的 DNA 修复能力，并编码一种必需的组蛋白样蛋白 DrHU。
我们旨在研究来自耐辐射球菌的组蛋白样 HU 蛋白的磷酸化调控作用。
利用 LC-MS/MS 分析确定内源性 DrHU 的磷酸化位点。使用同源建模（Swissmodel）根据金黄色葡萄球菌 HU-DNA 结构（PDB ID：4QJU）作为起始模型，预测 DrHU-DNA 的结构。生成两种类型的突变蛋白 T37E 和 T37A 以探索它们的 DNA 结合亲和力。使用互补敲除策略生成 DrHU 缺失株/ pk-T37A 和 DrHU 缺失株/ pk-T37E 进行生长曲线和表型分析。
在大多数细菌 HU 蛋白中存在的磷酸化位点 Thr37 位于 DrHU 的假定蛋白-DNA 相互作用界面上。与野生型蛋白相比，将该苏氨酸替换为模拟磷酸化永久状态的谷氨酸的突变蛋白（T37E）显示出增强的双链 DNA 结合能力，但对羟基自由基切割的保护作用减弱。在 DrHU 缺失株中互补 T37E 导致生长缺陷，并使细胞对 UV 辐射和氧化应激敏感。
磷酸化调节来自 D. radiodurans 的组蛋白样 HU 蛋白的 DNA 结合能力，这有助于该生物体的环境适应。
