Kinetic Instability of p53 Core Domain Mutants: IMPLICATIONS FOR RESCUE BY SMALL MOLECULES

Assaf Friedler,Dmitry B Veprintsev,Lars O Hansson,Alan R Fersht

doi:10.1074/jbc.m302458200

Assaf Friedler, Dmitry B Veprintsev + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m302458200

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Abstract

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain. Many of these mutants are thermodynamically unstable at body temperature. Here we show that these mutants also denature within minutes at 37 degrees C. The half-life (t(1/2)) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increasing thermodynamic instability. The highly destabilized mutant I195T had a t(1/2) of less than 1 min. The wild-type p53-(94-360) construct, containing the core and tetramerization domains, was more stable, with t(1/2) = 37 min at 37 degrees C, similar to full-length p53. After unfolding, the denatured proteins aggregated, the rate increasing with higher concentrations of protein. A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain. Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it.

Highlights

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain
We have recently demonstrated the feasibility of this approach and developed a peptide, FL-CDB3, which is able to stabilize mutant p53 core domain and rescue its sequence-specific DNA binding activity (8)
We find that p53 core domain mutants are kinetically unstable, with an in vitro half-life of only a few minutes at 37 °C

Summary

IMPLICATIONS FOR RESCUE BY SMALL MOLECULES*

A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. We conclude that peptides like FL-CDB3,1 which rescue the conformation of destabilized p53 mutants, must act during or immediately after biosynthesis and maintain the mutant protein in its native state until it reaches the nucleus and binds its sequence-specific DNA. Missense point mutations in p53 (see the mutation data base in www.iacr.fr/p53) lead to its inactivation and are involved in many human cancers (3, 4) These are mapped mainly to the DNA-binding core domain (5).

EXPERIMENTAL PROCEDURES

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DISCUSSION