Solution structure of p53 core domain: Structural basis for its instability

Abstract

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using stateof-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal͞mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.dynamics ͉ stability ͉ protein ͉ NMR ͉ isotope labeling

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs

“…The crystal structure of T-p53C-G245S revealed distinct structural changes in the immediate environment of the mutation site. Observed conformational changes in some surface loops (e.g., in the L1 loop and the S7-S8 loop) can be attributed to differences in crystallization conditions and crystal packing for T-p53C and T-p53C-G245S and ref lect the intrinsic conformational f lexibility of these loop regions (12,17,19). In T-p53C-G245S, the hydroxyl group of Ser-245 points toward the zinc ligands Cys-238 and Cys-242 and displaces a structural water molecule that is observed in the wild-type and various mutant structures (Fig.…”

“…There is growing evidence that p53 has evolved to be highly dynamic and intrinsically unstable (12,19,39). As a negative side effect of this evolutionary process, the core domain of p53 has in a way become more susceptible to cancer-associated mutations in the ␤-sandwich.…”

“…Most p53 cancer mutations are located in the DNA-binding core domain of the protein (3). This domain has been structurally characterized in complex with its cognate DNA by x-ray crystallography (5,10,11) and in its free form in solution by NMR (12). It consists of a central ␤-sandwich that serves as a basic scaffold for the DNA-binding surface.…”

Reactivation of mutant p53: molecular mechanisms and therapeutic potential

“…This notion is supported by recent studies of the solution structure of the p53 core domain that allowed the identification of several structural elements conferring flexibility to the core domain. This suggests that p53 has evolved to be dynamic and conformationally unstable (Canadillas et al, 2006).…”

Quaternary structures of tumor suppressor p53 and a specific p53–DNA complex

“…The structures of the core domain complexes with DNA have been solved by crystallography (15)(16)(17), and in solution in the absence of DNA by NMR (18). The structure of the tetramerization domain has been solved by both NMR and x-ray crystallography (19-21).Structural studies on full-length p53 have been impeded both by its intrinsic instability and the presence of disordered regions (8,18,22,23). There is an increasing number of proteins being discovered to have globular domains linked by intrinsically disordered regions (24), and so the determination of such structures will be a recurring problem.…”