Cooperative binding of tetrameric p53 to DNA
Cooperative binding of tetrameric p53 to DNA
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DOI：
10.1016/j.jmb.2004.06.071
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发表时间：
2004-08-27
影响因子：
5.6
通讯作者：
Fersht, AR
中科院分区：
文献类型：
--
作者：
Weinberg, RL;Veprintsev, DB;Fersht, AR
我们通过分析性超离心和荧光各向异性分析了p53截短突变体与序列特异性DNA的结合。合成的30个碱基对的DNA寡聚体含有p53的20个碱基对识别元件，由每个p53单体5个碱基对的4个位点组成。我们发现在低离子强度下的结合被非特异性结合的伪影所掩盖，因此在较高离子强度下进行测量。构建体p53 CT（残基94-360，含有DNA结合核心和四聚体结构域）的分析超离心得到其二聚体四聚体平衡的解离常数为-3 μ M，与全长蛋白质的解离常数相似。超离心和荧光各向异性分析表明，p53 CT与DNA构建体形成了2：1化学计量比的复合物（二聚体：DNA）。p53 CT（1-100 nm范围）与DNA的结合是高度合作的，希尔系数为1.8（二聚体：DNA）。p53 CT的二聚体L344 A突变体具有受损的四聚化。它与全长DNA p53识别序列结合，但亲和力比野生型蛋白低6倍。它没有与含有两个特定的五个碱基对位点和两个随机位点的30聚体DNA构建体形成可检测的复合物，强调了结合的高协同性。p53的基本活性单位似乎是由DNA结合诱导的四聚体，尽管它在低浓度下是二聚体。(C)2004爱思唯尔有限公司保留所有权利。
We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of -3 muM for its dimertetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations. (C) 2004 Elsevier Ltd. All rights reserved.