Wright Jon D, Noskov Sergey Yu, Lim Carmay
Institute of Biomedical Sciences, Academia Sinica, 11529 Taipei, Taiwan.
Nucleic Acids Res. 2002 Apr 1;30(7):1563-74. doi: 10.1093/nar/30.7.1563.
The mutation of R273-->H in the p53 core domain (p53-CD) is one of the most common mutations found in human cancers. Although the 273H p53-CD retains the wild-type conformation and stability, it lacks sequence-specific DNA binding, a transactivation function and growth suppression. However, mutating T284-->R in the 273H p53-CD restores the DNA binding affinity, and transactivation and tumour suppressor functions. Since X-ray/NMR structures of DNA-free or DNA-bound mutant p53-CD molecules are unavailable, the factors governing the loss and rescue of sequence-specific DNA binding in the 273H and 273H+284R p53-CD, respectively, are unclear. Hence, we have carried out molecular dynamics (MD) simulations of the wild-type, single mutant and double mutant p53-CD, free and DNA bound, in the presence of explicit water molecules. Based on the MD structures, the DNA-binding free energy of each p53 molecule has been computed and decomposed into component energies and contributions from the interface residues. The wild-type and mutant p53-CD MD structures were found to be consistent with the antibody-binding, X-ray and NMR data. The predicted DNA binding affinity and specificity of both mutant p53-CDs were also in accord with experimental data. The non-detectable DNA binding of the 273H p53-CD is due mainly to the disruption of a hydrogen-bonding network involving R273, D281 and R280, leading to a loss of major groove binding by R280 and K120. The restoration of DNA binding affinity and specificity of the 273H+284R p53-CD is due mainly to the introduction of another DNA-binding site at position 284, leading to a recovery of major groove binding by R280 and K120. The important role of water molecules and the DNA major groove conformation as well as implications for structure-based linker rescue of the 273H p53-CD DNA-binding affinity are discussed.
p53核心结构域（p53-CD）中R273→H的突变是人类癌症中最常见的突变之一。尽管273H p53-CD保留了野生型构象和稳定性，但它缺乏序列特异性DNA结合、反式激活功能和生长抑制作用。然而，在273H p53-CD中将T284→R突变可恢复DNA结合亲和力、反式激活和肿瘤抑制功能。由于尚无无DNA或与DNA结合的突变型p53-CD分子的X射线/核磁共振结构，因此分别控制273H和273H+284R p53-CD中序列特异性DNA结合丧失和恢复的因素尚不清楚。因此，我们在存在明确水分子的情况下，对野生型、单突变和双突变p53-CD进行了自由态和与DNA结合态的分子动力学（MD）模拟。基于MD结构，计算了每个p53分子的DNA结合自由能，并将其分解为组成能量和来自界面残基的贡献。发现野生型和突变型p53-CD的MD结构与抗体结合、X射线和核磁共振数据一致。两种突变型p53-CD的预测DNA结合亲和力和特异性也与实验数据相符。273H p53-CD无法检测到DNA结合主要是由于涉及R273、D281和R280的氢键网络被破坏，导致R280和K120失去对大沟的结合。273H+284R p53-CD的DNA结合亲和力和特异性的恢复主要是由于在284位引入了另一个DNA结合位点，导致R280和K120恢复对大沟的结合。讨论了水分子和DNA大沟构象的重要作用以及对基于结构的恢复273H p53-CD DNA结合亲和力的影响。
