Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.
J Biol Chem. 2010 Jan 22;285(4):2857-66. doi: 10.1074/jbc.M109.075861. Epub 2009 Nov 17.
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.
p53 是一种转录因子，可维持基因组完整性，其功能在 50%的人类癌症中丧失。p53 的大多数突变集中在核心结构域内。在这里，我们研究了低 pH 值对野生型 (wt) p53 核心结构域 (p53C) 和 R248Q 突变体的结构的影响。在低 pH 值下，色氨酸残基部分暴露于溶剂中，表明三级结构不稳定。另一方面，圆二色性测定表明二级结构增加。探针 bis-ANS（双-8-苯胺基萘-1-磺酸盐）的结合表明，wt 和突变体 p53C 在低 pH 值时，疏水性口袋的暴露增加。这种行为伴随着脲变性下的协同性缺乏和酸性 pH 值下 p53C 稳定性下降。综上所述，这些结果表明 p53C 在低 pH 值（5.0）下获得部分展开的构象（熔融球状态）。这种构象的流体力学性质介于天然和变性构象之间。与 pH 值 7.2 相比，（1）H-（15）N HSQC NMR 光谱证实该蛋白质在酸性 pH 值下具有典型的熔融球结构。在培养的人乳腺癌细胞（MCF-7）中转染 p53-GFP 后，发现 p53 定位于酸性小泡中，这表明低 pH 值构象存在于细胞中。低 pH 值应激也倾向于使细胞中 p53 水平升高。综上所述，所有这些数据表明 p53 可能在酸性微环境中发挥生理或病理作用。
