Davison T S, Nie X, Ma W, Lin Y, Kay C, Benchimol S, Arrowsmith C H
Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada.
J Mol Biol. 2001 Mar 23;307(2):605-17. doi: 10.1006/jmbi.2001.4450.
P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.
P53是一种同四聚体肿瘤抑制蛋白，参与调控细胞增殖和死亡基因的转录控制。为了探究寡聚化在这一功能中所起的作用，我们之前设计并表征了一系列p53蛋白，这些蛋白通过对正常四聚体寡聚化结构域中的Met340或Leu344残基进行亲水性取代，从而改变了寡聚状态。尽管此类突变对寡聚化结构域的整体二级结构含量影响不大，但相对于野生型结构域，其溶解度和对热变性的抗性均大幅降低。在此，我们报告了一种双突变p53的设计与表征，该双突变体在Met340和Leu344位点的残基发生了改变。双突变Met340Glu/Leu344Lys和Met340Gln/Leu344Arg产生了该蛋白不同的二聚体形式。此外，我们通过核磁共振结构测定验证了双突变体Met340Gln/Leu344Arg本质上是一个“半四聚体”。对全长p53寡聚突变体的体内活性分析表明，虽然细胞周期阻滞需要四聚体p53，但单体和二聚体的转录反式激活活性分别大致保留背景活性和野生型活性的一半。
