Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue.
Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue.
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DOI：
10.1107/s0907444913020830
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发表时间：
2013-10
期刊：
影响因子：
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通讯作者：
B. Wallentine;Ying Wang;V. Tretyachenko-Ladokhina;M. Tan;D. F. Senear;H. Luecke
中科院分区：
文献类型：
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作者：
B. Wallentine;Ying Wang;V. Tretyachenko-Ladokhina;M. Tan;D. F. Senear;H. Luecke
为了深入了解某些第二位点抑制突变拯救肿瘤抑制蛋白p53的大量癌症突变的功能的机制，确定了四种p53核心结构域变体的X射线晶体学结构。其中包括一个致癌突变体V157 F，两个单位点抑制突变体N235 K和N239 Y，以及拯救的癌症突变体V157 F/N235 K/N239 Y。V157 F突变在疏水核心的链S4内用较大的疏水苯丙氨酸取代较小的疏水缬氨酸。这种癌症突变体的结构显示在p53核心结构域的整体折叠中没有总体结构变化，仅在蛋白质的疏水核心内侧链的微小重排。根据生化分析，这些小的局部扰动会引起蛋白质的不稳定性，使自由能增加3.6 kcal mol（-1）（15.1 kJ mol（-1））。进一步的生物化学证据表明，每个抑制突变，N235 K或N239 Y，单独作用，以恢复热力学稳定V157 F，两者一起比单独更有效。当在允许温度下评估时，发现所有获救的突变体具有野生型DNA结合活性，从而指出热力学稳定性是关键的潜在变量。有趣的是，热力学分析表明，虽然N239 Y证明了野生型p53核心结构域的稳定性，但N235 K却没有。这些观察结果表明，不同的结构机制的救援。在N235 K和拯救的癌症突变体结构中发现的Lys 235和Glu 198之间的新盐桥表明依赖于稳定β-夹心支架的拯救机制。另一方面，取代N239 Y在该酪氨酸的芳环和相邻的Leu 137之间产生有利的疏水接触。令人惊讶的是，与野生型p53相比，获救的癌症突变体显示出比单独的癌症突变体大得多的结构偏差。这些抑制突变似乎通过产生稳定核心结构域的新型结构域内相互作用来拯救p53功能，从而补偿不稳定的V157 F突变。
To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol(-1) (15.1 kJ mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.