Ultraslow oligomerization equilibria of p53 and its implications
Ultraslow oligomerization equilibria of p53 and its implications
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DOI：
10.1073/pnas.0907840106
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发表时间：
2009-08-25
影响因子：
11.1
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
--
作者：
Natan, Eviatar;Hirschberg, Daniel;Fersht, Alan R.
肿瘤抑制因子p53在二聚体和四聚体之间的细胞浓度处于平衡。致癌突变型p53（mut）对癌细胞中p53野生型（wt）和mut等位基因的共表达产生显性负效应。据信，wt和mut经由其四聚化结构域形成活性减弱的异源四聚体。使用电喷雾质谱同位素标记的样品，我们直接测量的组成和反应元件DNA的存在和不存在下的p53复合物的形成率。四聚体的解离在37 ℃下出乎意料地非常慢（t（1/2）= 40 min），与二聚体的缓慢缔合相匹配，其比wt p53自发变性的半衰期长约4倍。在将wt四聚体与低DNA亲和力的致癌接触突变体R273 H混合时，我们观察到在细胞时间尺度上仅wt（4）、wt（2）mut（2）和mut（4）以1：2：1的比例缓慢形成。当wt和mut与响应元件DNA P21和BAX混合时，我们仅观察到复合物wt（4）.DNA、wt（2）mut（2）.DNA和mut（4）.DNA，其相对解离常数分别为1：4：71和1：13：85，解释了亲和力减弱的显性-负效应。p53二聚体在与应答元件DNA结合时迅速组装成四聚体，这是由p53 DNA结合结构域引发的。游离p53的缓慢寡聚化，与自发变性竞争，具有影响四聚动力学以及平衡的结合蛋白和DNA对p53的可能调节的影响。
The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wildtype (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t(1/2) = 40 min) at 37 degrees C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt(4), wt(2)mut(2), and mut(4), in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt(4).DNA, wt(2)mut(2).DNA, and mut(4).DNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.