Kinetic instability of p53 core domain mutants: implications for rescue by small molecules.
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.
Friedler
Assaf
A
Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Veprintsev
Dmitry B
DB
Hansson
Lars O
LO
Fersht
Alan R
AR
D019281:Dimerization; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics*
D009842:Oligopeptides; D016159:Tumor Suppressor Protein p53