Stabilising the DNA-binding domain of p53 by rational design of its hydrophobic core

被引:22
作者
Khoo, Kian Hoe [1 ]
Joerger, Andreas C. [1 ]
Freund, Stefan M. V. [1 ]
Fersht, Alan R. [1 ]
机构
[1] MRC Ctr Prot Engn, Cambridge CB2 0QH, England
基金
英国医学研究理事会;
关键词
drug design; folding intermediate; p53; protein stability; structure; TUMOR-SUPPRESSOR P53; THERMODYNAMIC STABILITY; STRUCTURAL BASIS; RESCUE; MUTANT; P63; P73; INTERMEDIATE; MUTATIONS; MECHANISM;
D O I
10.1093/protein/gzp018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and also low kinetic stability, which leads to rapid irreversible denaturation. Some oncogenic mutations of p53 act by just making the core domain thermosensitive, and so it is the target of novel anti-cancer drugs that bind to and stabilise the protein. Increasing the stability of the unstable core domain has also been crucial for biophysical and structural studies, in which a stabilised quadruple mutant (QM) is currently used. We generated an even more stabilised hexamutant (HM) by making two additional substitutions, Y236F and T253I, to the QM. The residues are found in the more stable paralogs p63 and p73 and stabilise the wild-type p53 core domain. We solved the structure of the HM core domain by X-ray crystallography at 1.75 A resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the beta-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 37 degrees C. Anomalies in biophysics and spectroscopy in urea-mediated denaturation curves of HM implied the accumulation of a folding intermediate, which may be related to those detected in kinetic experiments. The two additional mutations over-stabilise an unfolding intermediate. These results should be taken into consideration in drug design strategies for increasing the stability of temperature-sensitive mutants of p53.
引用
收藏
页码:421 / 430
页数:10
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