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PEDS Advance Access published online on June 10, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp018
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

Kian Hoe Khoo{dagger}, Andreas C. Joerger{dagger}, Stefan M.V. Freund and Alan R. Fersht1

MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, UK

1 To whom correspondence should be addressed. E-mail: arf25{at}cam.ac.uk

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 Å resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the β-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 37°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.

Keywords: drug design/folding intermediate/p53/protein stability/structure

Received May 5, 2009; revised May 5, 2009; accepted May 6, 2009.

{dagger} These authors contributed equally.


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K. H. Khoo, S. Mayer, and A. R. Fersht
Effects of Stability on the Biological Function of p53
J. Biol. Chem., November 6, 2009; 284(45): 30974 - 30980.
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