Specific sequences from the carboxyl terminus of human p53 gene product form anti-parallel tetramers in solution.

Abstract

Human p53 is a tumor-suppressor gene product associated with control of the cell cycle and with growth suppression, and it is known to form homotetramers in solution. To investigate the relationship of structure to tetramerization, nine peptides corresponding to carboxyl-terminal sequences in human p53 were chemically synthesized, and their equilibrium associative properties were determined by analytical ultracentrifugation. Secondary structure, as determined by circular dichroism measurements, was correlated with oligomerization properties of each peptide. The sedimentation profiles of peptides 319-393 and 319-360 fit a two-state model of peptide monomers in equilibrium with peptide tetramers. Successive deletion of amino-and carboxyl-terminal residues from 319-360 reduced tetramer formation. Further, substitution of alanine for Leu-323, Tyr-327, and Leu-330 abolished tetramerization. Circular dichroism studies showed that peptide 319-351 had the highest a-helix content, while the other peptides that did not form tetramers had low helical structure. These studies define a minimal region and identify certain critical residues involved in tetramerization. Cross-linking studies between monomer units in the tetramer suggest that the helices adopt an anti-parallel arrangement. We propose that conformational shifts in the helical structure of the p53 tetramerization domain result in a repositioning of subunits relative to one another. This repositioning provides an explanation relating conformational changes at the carboxyl terminus with changes in sequence-specific DNA binding by the highly conserved central domain.The p53 gene product is a phosphoprotein which has been implicated in the regulation of cell proliferation. Functional inactivation of the protein either by association with viral proteins (1) or, more frequently, by point mutations, as found in many human tumors, leads to cellular transformation (2). Wild-type p53 exhibits trans-activation of transcriptional activity which is thought to mediate its anti-proliferation activity (3). At the biological level, p53 undergoes oligomerization, and it can adopt two distinct conformational states, with low or high affinity for site-specific DNA binding (4). Many of the p53 mutants associated with human tumors appear to be incapable of binding DNA and can be distinguished from wild-type p53 by their oligomerization state and by their "mutant" immunoreactivity (5-7).Recent structure-function analyses of p53 have mapped the trans-activation domain to the 42 amino (N)-terminal amino acids (8). This region is highly susceptible to proteolytic digestion and may be loosely folded in the absence of ligand (9). Deletion analysis has shown that residues 90-286 form a stable domain which binds DNA with specificity (4).Subtilisin treatment of the p53 protein generates a 191-amino acid protease-resistant monomeric fragment from the midsection that corresponds to a highly conserved region in which the majority of missense mutations occur (9, 10).We have a limited unders...

Calcium-dependent Interaction of S100B with the C-terminal Domain of the Tumor Suppressor p53

“…The YF-O2 peptide on p53 is involved in the dimerization and in subsequent tetramerization of p53 dimers (28,(35)(36)(37). The K d value found for the tetramer-monomer transition of the C-terminal domain of p53 was determined to be 1-10 M (28, 35).…”

“…The minimal domain required for cell transformation is in bold (44). The oligomerization (28) and cytoplasmic sequestration domain is boxed (27). The putative PKC phosphorylation sites and PAb421 epitope are indicated (30).…”

“…319 -393 peptide was synthesized as described previously (28). The peptide was dissolved in 20 mM HEPES-NaOH, pH 7.5, at the concentration of 98 M. Other peptides were synthesized by the solid phase method, using Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry on an Applied Biosystems 430A peptide synthesizer.…”

Unfolding, Aggregation, and Amyloid Formation by the Tetramerization Domain from Mutant p53 Associated with Lung Cancer

“…Under these conditions, the WT peptide exhibited double minimum bands at 208 and 222 nm, which is a characteristic of the p53 tetramer structure (30). The helicity of the WT peptide reached a plateau at 10 μM peptide concentration with negative [θ] 222 values.…”

DNA damage activates p53 through a phosphorylation–acetylation cascade

“…We also prepared peptides with alanines replacing residues )AAA] in the tetramerization domain; these substitutions prevent the formation of p53 oligomers (Sakamoto et al 1994). For p300, phosphorylation at either site had no effect on acetylation, and the monomer form of the carboxy-terminal fragment also was acetylated well by p300 (Fig.…”