Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation

Abstract

The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely (ΔT(m) = 4.8 ∼ -46.8 °C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.

FIGURE 1.

Amino acid sequences for the WT and mutant p53TD peptides. A, amino acid sequences and the positions of the missense mutations in the TD of p53; β-strand residues are highlighted in red, α-helical residues are highlighted in blue. B, space-filling model of p53TD (Protein Data Bank code 3SAK) prepared with MolFeat (version 4.0, FiatLux Corp.) The amino acid residues of the mutation site in the p53TD and the location of these residues in the tetrameric structure are shown. The primary dimers are depicted, and the other dimer is removed to give a direct view of the interior of the protein. The right dimer was obtained by rotating the structure in the left picture by 180° around the horizontal axis.

FIGURE 2.

Thermal denaturation of WT and mutant p53TD peptides. Thermal denaturation of the peptides was analyzed by measuring the ellipticity at 222 nm for peptide solutions containing 10 μm peptide in 50 mm sodium phosphate, pH 7.5, 100 mm NaCl over the range of 4 to 96 °C, with a scan rate of 1 °C per minute.

FIGURE 3.

Fraction of tetramer at 37 °C at concentrations of 13 nm (endogenous p53 level in unstressed cell) and 36 nm (stressed cell) against the value of K_d. Each data point represents the value of a mutant at 13 nm (solid circles) and 36 nm (open circles). The fraction of tetramer at each concentration was calculated from the dissociation constant given in Table 1 assuming a two-state monomer-tetramer model (31). Monomer mutants 11, 12, 25, 32, and 35, and dimer mutants 30, 36, and 39 are not shown.