Folding of tetrameric p53: oligomerization and tumorigenic mutations induce misfolding and loss of function.
ABSTRACT The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 degrees C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.
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ABSTRACT: Large-scale tumor sequencing projects are now underway to identify genetic mutations that drive tumor initiation and development. Most studies take a gene-based approach to identifying driver mutations, highlighting genes mutated in a large percentage of tumor samples as those likely to contain driver mutations. However, this gene-based approach usually does not consider the position of the mutation within the gene or the functional context the position of the mutation provides. Here we introduce a novel method for mapping mutations to distinct protein domains, not just individual genes, in which they occur, thus providing the functional context for how the mutation contributes to disease. Furthermore, aggregating mutations from all genes containing a specific protein domain enables the identification of mutations that are rare at the gene level, but that occur frequently within the specified domain. These highly mutated domains potentially reveal disruptions of protein function necessary for cancer development. We mapped somatic mutations from the protein coding regions of 100 colon adenocarcinoma tumor samples to the genes and protein domains in which they occurred, and constructed topographical maps to depict the "mutational landscapes" of gene and domain mutation frequencies. We found significant mutation frequency in a number of genes previously known to be somatically mutated in colon cancer patients including APC, TP53 and KRAS. In addition, we found significant mutation frequency within specific domains located in these genes, as well as within other domains contained in genes having low mutation frequencies. These domain "peaks" were enriched with functions important to cancer development including kinase activity, DNA binding and repair, and signal transduction. Using our method to create the domain landscapes of mutations in colon cancer, we were able to identify somatic mutations with high potential to drive cancer development. Interestingly, the majority of the genes involved have a low mutation frequency. Therefore, the method shows good potential for identifying rare driver mutations in current, large-scale tumor sequencing projects. In addition, mapping mutations to specific domains provides the necessary functional context for understanding how the mutations contribute to the disease, and may reveal novel or more refined gene and domain target regions for drug development.BMC Genomics 01/2012; 13 Suppl 4:S9. · 4.07 Impact Factor
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ABSTRACT: Aggregation of p53 is initiated by first-order processes that generate an aggregation-prone state with parallel pathways of major or partial unfolding. Here, we elaborate the mechanism and explore its consequences, beginning with the core domain and extending to the full-length p53 mutant Y220C. Production of large light-scattering particles was slower than formation of the Thioflavin T-binding state and simultaneous depletion of monomer. EDTA removes Zn(2+) to generate apo-p53, which aggregated faster than holo-p53. Apo-Y220C also aggregated by both partial and major unfolding. Apo-p53 was not an obligatory intermediate in the aggregation of holo-p53, but affords a parallel pathway that may be relevant to oncogenic mutants with impaired Zn(2+) binding. Full-length tetrameric Y220C formed the Thioflavin T-binding state with similar rate constants to those of core domain, consistent with a unimolecular initiation that is unaffected by neighboring subunits, but very slowly formed small light-scattering particles. Apo-Y220C and aggregated holo-Y220C had little, if any, seeding effect on the initial polymerization of holo-Y220C (measured by Thioflavin T binding), consistent with initiation being a unimolecular process. But apo-Y220C and aggregated holo-Y220C accelerated somewhat the subsequent formation of light-scattering particles from holo-protein, implying coaggregation. The implications for cancer cells containing wild-type and unstable mutant alleles are that aggregation of wild-type p53 (or homologs) might not be seeded by aggregated mutant, but it could coaggregate with p53 or other cellular proteins that have undergone the first steps of aggregation and speed up the formation of microscopically observable aggregates.Proceedings of the National Academy of Sciences 08/2012; 109(34):13590-5. · 9.68 Impact Factor
Article: Molecular Dynamic Simulation Insights into the Normal State and Restoration of p53 Function.[show abstract] [hide abstract]
ABSTRACT: As a tumor suppressor protein, p53 plays a crucial role in the cell cycle and in cancer prevention. Almost 50 percent of all human malignant tumors are closely related to a deletion or mutation in p53. The activity of p53 is inhibited by over-active celluar antagonists, especially by the over-expression of the negative regulators MDM2 and MDMX. Protein-protein interactions, or post-translational modifications of the C-terminal negative regulatory domain of p53, also regulate its tumor suppressor activity. Restoration of p53 function through peptide and small molecular inhibitors has become a promising strategy for novel anti-cancer drug design and development. Molecular dynamics simulations have been extensively applied to investigate the conformation changes of p53 induced by protein-protein interactions and protein-ligand interactions, including peptide and small molecular inhibitors. This review focuses on the latest MD simulation research, to provide an overview of the current understanding of interactions between p53 and its partners at an atomic level.International Journal of Molecular Sciences 01/2012; 13(8):9709-40. · 2.60 Impact Factor