Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
Center for Drug Discovery and Innovation, University of South Florida, Tampa, FL 33612, USA.
Biomolecules. 2019 Mar 2;9(3):83. doi: 10.3390/biom9030083.
The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3⁻5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.
紊乱的 p53 转录激活结构域（p53TAD）含有特定水平的瞬态螺旋二级结构，这对于其与负调节剂，即鼠双微体 2（Mdm2）和 MdmX 的结合是必需的。p53 与 Mdm2 和 MdmX 的相互作用也受到 p53TAD 的翻译后修饰（PTMs）的调节，包括 S15、T18 和 S20 的磷酸化，这抑制了 p53-Mdm2 的结合。目前尚不清楚 p53TAD 中的瞬态二级结构水平是否会因磷酸化或其他 PTM 而改变。我们使用磷酸模拟突变体来确定在位置 15 和 18 处添加负电荷是否会对 p53TAD 的瞬态二级结构和蛋白质-蛋白质结合产生任何影响。我们使用组合的生物物理和结构方法，研究了单磷酸模拟和多磷酸模拟对 p53TAD 的瞬态二级结构及其与 Mdm2、MdmX 和 KIX 结构域的相互作用的影响。磷酸模拟物将 Mdm2 和 MdmX 的结合亲和力降低了 3-5 倍，但对瞬态二级结构的变化很小，这表明磷酸化对 p53TAD-Mdm2 相互作用的去稳定作用主要是静电的。磷酸模拟物对 p53-KIX 相互作用没有影响，这表明磷酸化 p53 与 KIX 的结合增加可能受到其负调节剂竞争减少的影响。
