Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain.

The p53 tumor suppressor oligomerization domain, a dimer of two primary dimers, is an independently folding domain whose subunits consist of a beta-strand, a tight turn and an alpha-helix. To evaluate the effect of hydrophobic side-chains on three-dimensional structure, we substituted residues Phe341 and Leu344 in the alpha-helix with other hydrophobic amino acids. Substitutions that resulted in residue 341 having a smaller side-chain than residue 344 switched the stoichiometry of the domain from tetrameric to dimeric. The three-dimensional structure of one such dimer was determined by multidimensional NMR spectroscopy. When compared with the primary dimer of the wild-type p53 oligomerization domain, the mutant dimer showed a switch in alpha-helical packing from anti-parallel to parallel and rotation of the alpha-helices relative to the beta-strands. Hydrophobic side-chain size is therefore an important determinant of a protein fold.

Structure-based rescue of common tumor-derived p53 mutants.

The p53 tumor suppressor protein induces cell-cycle arrest or cell death in response to DNA-damaging agents, such as radiation and many of the chemotherapeutics used in cancer therapy. The function of p53 is dependent on its ability to bind DNA in a sequence-specific manner, but in one-half of all human tumors, its sequence-specific DNA binding domain is compromised by single-amino acid substitutions. The nature of these substitutions, which target residues that directly contact DNA or that stabilize the structure of the DNA binding domain, has raised concerns as to whether the function of p53 mutants could ever be rescued. Nevertheless, pharmaceuticals that restore function to p53 mutants could specifically suppress proliferation of cancer cells in patients. To determine whether tumor-derived p53 mutants are irreversibly inactivated, we introduced basic residues in their DNA binding domains, aiming to establish novel contacts between p53 and the DNA phosphate backbone. In three of the seven most common p53 mutants, replacement of Thr284 with Arg significantly enhanced DNA binding affinity, without affecting DNA binding specificity, and rescued their transactivation and tumor suppressor functions. Thus, many tumor-derived p53 mutants retain their sequence-specific DNA binding determinants and can be activated to suppress tumor growth.