Dornan David, Shimizu Harumi, Burch Lindsay, Smith Amanda J, Hupp Ted R
Cancer Research UK Laboratories, P53 Signal Transduction Group, Department of Molecular and Cellular Pathology, University of Dundee, Dundee DD1 9SY, United Kingdom.
Mol Cell Biol. 2003 Dec;23(23):8846-61. doi: 10.1128/MCB.23.23.8846-8861.2003.
The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that proline-directed acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.
转录共激活因子p300无法使天然的p53四聚体发生乙酰化，从而揭示了p300催化的乙酰化存在内在构象限制。共有序列位点DNA是一种变构效应物，可促进p53的乙酰化，这表明p300在p53四聚体内具有一个未明确的构象灵活界面。为了鉴定这种构象响应性的p300结合位点，利用噬菌体肽展示文库对p300进行肽段筛选，该技术能够确定新的蛋白质-蛋白质界面。富集的p300结合肽含有脯氨酸重复（PXXP/PXPXP）基序，实际上p53反式激活结构域内存在五个脯氨酸重复基序，这表明p53的该区域可能含有第二个p300接触位点。p300在体外与源自脯氨酸重复结构域的含PXXP肽结合，且含PXXP肽抑制p53的序列特异性DNA依赖性乙酰化，这表明p53乙酰化需要p300与p53中的LXXLL和相邻的PXXP基序对接。p53脯氨酸重复基序的缺失通过阻止p300进入p53-DNA复合物而防止p53的DNA依赖性乙酰化。序列特异性DNA对脯氨酸重复结构域在体内驱动p53乙酰化提出了绝对要求。染色质免疫沉淀用于表明脯氨酸重复缺失突变体p53在体内与p21启动子结合，但未发生乙酰化，这表明p53的脯氨酸定向乙酰化是DNA结合后的事件。PXXP重复扩展了p300靶向反式激活结构域的基本界面，且启动子处p53的脯氨酸定向乙酰化表明p300介导的乙酰化在体内可能受到底物构象的高度限制。
