Kinetic Instability of p53 Core Domain Mutants

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 °C. The half-life (t1 ⁄2) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increas-ing thermodynamic instability. The highly destabilized mutant I195T had a t1 ⁄2 of less than 1 min. The wild-type p53-(94 –360) construct, containing the core and tet-ramerization domains, was more stable, with t1 ⁄2 ‫؍‬ 37 min at 37 °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 unsta-ble mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggre-gation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it. The tumor suppressor protein p53 is at the center of a net-work of many proteins whose role is to guard the cell from potential cancer (1, 2). Missense point mutations in p53 (see the mutation data base in www.iacr.fr/p53) lead to its inacti-vation and are involved in many human cancers (3, 4). These are mapped mainly to the DNA-binding core domain (5). Classification of the core domain mutants according to their DNA binding and thermodynamic stability properties results in the following three major phenotypes (6): 1) DNA contact mutants, which are unable to bind DNA but are as stable and almost as folded as the wild-type; 2) locally distorted and/or weakly destabilized mutants, partly unfolded, which are not fully able to bind DNA and are usually destabilized by less than ϳ2 kcal/mol (6); and 3) globally destabilized mutants, which possess a high degree of unfolding and are destabilized by more than ϳ3 kcal/mol. Accordingly, one of the important targets in cancer therapy is the rescue of p53 mutants (4, 7–10). Small molecules that bind the native but not the denatured state can restore the activity of unfolded p53 mutants by shifting the equilibrium toward the native state (7–10). 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). Here, we have studied the kinetics of unfolding of p53 core domain mutants to gain a deeper understanding of the basis for their inactivation and the requirements for their rescue. We find that p53 core domain mutants are kinetically unstable, with an in vitro half-life of only a few minutes at 37 °C. We find a clear correlation between the thermodynamic and kinetic instability of p53 core domain mutants, the more unstable the mutant, the shorter the half-life. We also show that peptides can slow down the unfolding rate of the p53 core domain. We conclude that peptides like FL-CDB3, 1 which rescue the con-formation of destabilized p53 mutants, must act during or immediately after biosynthesis and maintain the mutant pro-tein in its native state until it reaches the nucleus and binds its sequence-specific DNA.