ABSTRACT
p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Multimerization , Protein Structure, Quaternary , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , DNA-Binding Proteins/genetics , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Structure, Secondary , Sequence Alignment , Thermodynamics , Trans-Activators/chemistry , Trans-Activators/metabolism , Tumor Protein p73 , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
One protein--p53--plays nemesis to most cancers by condemning damaged cells to death or quarantining them for repair. But the activity of p53 relies on its intact native conformation, which can be lost following mutation of a single nucleotide. With thousands of such mutations identified in patients, how can a future cancer drug buttress this fragile protein structure and restore the cell's natural defence?
Subject(s)
Mutation , Tumor Suppressor Protein p53/physiology , Animals , Benzamides , DNA/metabolism , Humans , Imatinib Mesylate , Piperazines/therapeutic use , Protein Folding , Pyrimidines/therapeutic use , Tumor Suppressor Protein p53/chemistryABSTRACT
The tumour suppressor p53 is mutated in half of all human cancers, most frequently with missense substitutions in its core domain. We present a new assessment of the mutation database based on quantitative folding and DNA-binding studies of the isolated core domain. Our data identify five distinct mutant classes that correlate with four well-defined regions of the core domain structure. On extrapolation to 37 degrees C the wild-type protein has a stability of 3.0 kcal/mol. This also emerges as an oncogenic threshold: all beta-sandwich mutants destabilized by this amount (50% denatured) are expected to promote cancer. Other weakly destabilizing mutations are restricted to loop 3 in the DNA-binding region. Drugs that stabilize mutant p53 folding have the potential to reactivate apoptotic signalling pathways in tumour cells either by transactivation-dependent or independent pathways. Using an affinity ligand as a proof of principle we have recovered the thermodynamic stability of the hotspot G245S. With reference states for the five mutant classes as a guide, future therapeutic strategies may similarly stabilize partially structured or binding states of mutant p53 that restore limited p53 pathways to tumour suppression.
Subject(s)
Genes, p53 , Mutation , Protein Folding , Tumor Suppressor Protein p53/chemistry , Apoptosis , Databases, Factual , Humans , Models, Chemical , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Denaturation , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Temperature , Thermodynamics , Transcriptional ActivationABSTRACT
Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn2+ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25 degrees C and 9.8 kcal/mol at 10 degrees C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.
