Factors governing loss and rescue of DNA binding upon single and double mutations in the p53 core domain

The mutation of R273→H in the p53 core domain (p53-CD) is one of the most common mutations found in human cancers. Although the 273H p53-CD retains the wild-type conformation and stability, it lacks sequence-specific DNA binding, a transactivation function and growth suppression. However, mutating T284→R in the 273H p53-CD restores the DNA binding affinity, and transactivation and tumour suppressor functions. Since X-ray/NMR structures of DNA-free or DNA-bound mutant p53-CD molecules are unavailable, the factors governing the loss and rescue of sequence-specific DNA binding in the 273H and 273H+284R p53-CD, respectively, are unclear. Hence, we have carried out molecular dynamics (MD) simulations of the wild-type, single mutant and double mutant p53-CD, free and DNA bound, in the presence of explicit water molecules. Based on the MD structures, the DNA-binding free energy of each p53 molecule has been computed and decomposed into component energies and contributions from the interface residues. The wild-type and mutant p53-CD MD structures were found to be consistent with the antibody-binding, X-ray and NMR data. The predicted DNA binding affinity and specificity of both mutant p53-CDs were also in accord with experimental data. The non-detectable DNA binding of the 273H p53-CD is due mainly to the disruption of a hydrogen-bonding network involving R273, D281 and R280, leading to a loss of major groove binding by R280 and K120. The restoration of DNA binding affinity and specificity of the 273H+284R p53-CD is due mainly to the introduction of another DNA-binding site at position 284, leading to a recovery of major groove binding by R280 and K120. The important role of water molecules and the DNA major groove conformation as well as implications for structure-based linker rescue of the 273H p53-CD DNA-binding affinity are discussed.     

Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study

The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H) in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R). In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R) can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis) to treat human cancer.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.     

Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

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 °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.     

Structures of p53 cancer mutants and mechanism of rescue by second-site suppressor mutations

We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the "structural" oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg(168) mimics the role of Arg(249) in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.     

Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA-binding domain, is one of the major hallmarks of cancer. The R273 position is a DNA-contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant variants of R273 in p53 have different survival rates, indicating that the DNA-contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild-type (WT) p53: [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical, and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but they also show distinct structural stability, aggregation, and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulfide mediated, leading to cross-β, thioflavin-T-positive aggregates, whereas hydrophobic interactions dominate self-assembly in [R273L]p53, leading to a mixture of amyloid and amorphous aggregates. Molecular dynamics simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process-specific protein aggregation as a target.     

Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion

The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.     

"Similarity trap" in protein-protein interactions could be carcinogenic: simulations of p53 core domain complexed with 53BP1 and BRCA1 BRCT domains

Similar binding sites often imply similar protein-protein interactions and similar functions; however, similar binding sites may also constitute traps for nonfunctional associations. How are similar sites distinguished to prevent misassociations? BRCT domains from breast cancer-susceptibility gene product BRCA1 and protein 53BP1 have similar structures yet different binding behaviors with p53 core domain. 53BP1-BRCT domain forms a stable complex with p53. In contrast, BRCA1-p53 interaction is weak or other mechanisms operate. To delineate the difference, we designed 13 BRCA1-BRCT mutants and computationally investigated the structural and stability changes compared to the experimental p53-53BP1 structure. Interestingly, of the 13, the 2 mutations that are cancerous and involve nonconserved residues are those that enforced p53 core domain binding with BRCA1-BRCT in a way similar to p53-53BP1 binding. Hence, falling into the "similarity trap" may disrupt normal BRCA1 and p53 functions. Our results illustrate how this trap is avoided in the native state.     

Probing potential binding modes of the p53 tetramer to DNA based on the symmetries encoded in p53 response elements

Symmetries in the p53 response-element (p53RE) encode binding modes for p53 tetramer to recognize DNA. We investigated the molecular mechanisms and biological implications of the possible binding modes. The probabilities evaluated with molecular dynamics simulations and DNA sequence analyses were found to be correlated, indicating that p53 tetramer models studied here are able to read DNA sequence information. The traditionally believed mode with four p53 monomers binding at all four DNA quarter-sites does not cause linear DNA to bend. Alternatively, p53 tetramer can use only two monomers to recognize DNA sequence and induce DNA bending. With an arrangement of dimer of AB dimer observed in p53 trimer–DNA complex crystal, p53 can recognize supercoiled DNA sequence-specifically by binding to quarter-sites one and four (H14 mode) and recognize Holliday junction geometry-specifically. Examining R273H mutation and p53–DNA interactions, we found that at least three R273H monomers are needed to disable the p53 tetramer, consistent with experiments. But just one R273H monomer may greatly shift the binding mode probabilities. Our work suggests that p53 needs balanced binding modes to maintain genome stability. Inverse repeat p53REs favor the H14 mode and direct repeat p53REs may have high possibilities of other modes.     

CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (B(max)) and its affinity (K(d)) for DNA. The compound, however, does not affect the affinity (K(d) value) of wild type p53 for DNA and only increases B(max) slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.     

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.