Butler James S, Loh Stewart N
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
Protein Sci. 2006 Nov;15(11):2457-65. doi: 10.1110/ps.062324206. Epub 2006 Sep 25.
p53 modulates a large number of cellular response pathways and is critical for the prevention of cancer. Wild-type p53, as well as tumorigenic mutants, exhibits the singular property of spontaneously losing DNA binding activity at 37 degrees C. To understand the molecular basis for this effect, we examine the folding mechanism of the p53 DNA binding domain (DBD) at elevated temperatures. Folding kinetics do not change appreciably from 5 degrees C to 35 degrees C. DBD therefore folds by the same two-channel mechanism at physiological temperature as it does at 10 degrees C. Unfolding rates, however, accelerate by 10,000-fold. Elevated temperatures thus dramatically increase the frequency of cycling between folded and unfolded states. The results suggest that function is lost because a fraction of molecules become trapped in misfolded conformations with each folding-unfolding cycle. In addition, at 37 degrees C, the equilibrium stabilities of the off-pathway species are predicted to rival that of the native state, particularly in the case of destabilized mutants. We propose that it is the presence of these misfolded species, which can aggregate in vitro and may be degraded in the cell, that leads to p53 inactivation.
p53调节大量细胞反应途径，对预防癌症至关重要。野生型p53以及致瘤突变体在37摄氏度时表现出自发丧失DNA结合活性的独特特性。为了解这种效应的分子基础，我们研究了p53 DNA结合结构域（DBD）在升高温度下的折叠机制。从5摄氏度到35摄氏度，折叠动力学没有明显变化。因此，DBD在生理温度下与在10摄氏度时一样，通过相同的双通道机制折叠。然而，解折叠速率加快了10000倍。升高的温度因此显著增加了折叠态与未折叠态之间循环的频率。结果表明，功能丧失是因为在每个折叠-解折叠循环中，一部分分子被困在错误折叠的构象中。此外，在37摄氏度时，预计偏离途径物种的平衡稳定性与天然状态相当，特别是在不稳定突变体的情况下。我们提出，正是这些错误折叠的物种的存在导致了p53失活，这些物种在体外可聚集，在细胞中可能被降解。
