Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration
Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration
复制标题
DOI：
10.1073/pnas.1105028108
复制
发表时间：
2011-05-17
影响因子：
11.1
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
--
作者：
Arbely, Eyal;Natan, Eviatar;Fersht, Alan R.
P53 DNA结合域(DBD)中的Lys120在DNA损伤时发生乙酰化。但是，乙酰化的作用和影响尚不清楚。我们通过体内掺入乙酰化赖氨酸制备了特异性地在Lys120，AcK120处乙酰化的P53，以研究乙酰化的生物物理和结构后果，这可能有助于阐明其生物学作用。在1.9埃分辨率下，乙酰化对DBD的整体晶体结构没有影响，但显著改变了盐浓度对DNA结合特异性的影响。在体外，p53在有效的生理盐浓度下随机结合DNA，直到较高的盐浓度才能与DNA特异性结合或区分其不同的反应元件。但是，在乙酰化方面，AcK120p53表现出特异性的DNA结合，并在有效生理盐浓度下区分不同的反应元件。AcK120P53和P53对同一DNA序列的亲和力最高，尽管乙酰化分别降低了第4位和第7位的共识C和G的重要性。结合DNA的P53和AcK120P3DBD的质谱图表明，它们优先分离成DNA(P53DBD)(4)或DNA(AcK120DBD)(4)的络合物，这表明不同的DBD偏爱不同的四元结构。这些结果与电子显微镜观察结果一致，即P53以不同于结合特定序列的松弛的四种状态与非特异性DNA结合。越来越多的证据表明，p53可以被随机DNA隔离，靶标搜索需要Lys120的乙酰化和/或与其他因素的相互作用，通过调节四元结构的变化来施加结合的特异性。
Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-angstrom resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNA(p53DBD)(4) or DNA (AcK120DBD)(4), indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.