Cesková Pavla, Chichger Havovi, Wallace Maura, Vojtesek Borek, Hupp Ted R
CRUK Cell Signaling Unit, University of Edinburgh, South Crewe Road, Edinburgh EH4 2XR, UK.
J Mol Biol. 2006 Mar 24;357(2):442-56. doi: 10.1016/j.jmb.2005.12.026. Epub 2005 Dec 27.
P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.
p53的乙酰化需要p300对接至激活域中的两个相邻位点，这继而介导四聚体的DNA依赖性乙酰化。为了进一步明确p53的DNA依赖性乙酰化机制，已用不同的p53亚型重建了一个体外系统，并用于揭示p53乙酰化的构象限制。从Sf9细胞中纯化的两种天然p53四聚体亚型，其C末端乙酰化位点内的磷酸化程度不同，二者均以序列特异性DNA依赖性方式被乙酰化。相比之下，从大肠杆菌表达系统中纯化的p53处于 largely denatured conformation，其乙酰化不依赖于DNA。加热天然p53以破坏折叠结构可恢复不依赖于DNA的乙酰化，类似于细菌表达的p53的情况。p53四聚体中至少有两个构象灵活性位点：第一个位于MDM2泛素化序列内的柔性S10β-折叠中，第二个位于C末端调节域中。因此，我们分析了DNA依赖性乙酰化是否与这两个区域中任一区域的构象变化相关。在S10β-折叠区域保持灵活性的条件下，低浓度的共有位点DNA以剂量依赖性方式维持p53的DNA依赖性乙酰化。促进乙酰化的寡核苷酸DNA刺激单克隆抗体PAb421和ICA-9的结合；这两种抗体的相邻表位与C末端乙酰化基序重叠。相比之下，使S10β-折叠无法与单克隆抗体DO-12结合并减弱单克隆抗体PAb421结合的弯曲寡核苷酸DNA可阻止乙酰化。这些数据表明，在没有DNA的情况下，p53的乙酰化基序处于隐蔽状态，但在DNA结合后，变构效应介导乙酰化基序的暴露，从而使四聚体进行DNA依赖性乙酰化。
