Common Conformational Effects in the P53 Protein of Vinyl Chloride-Induced Mutations

The tumor suppressor gene p53 has been identified as the most frequent site of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen, has been associated with specific A T transversions at codons 179, 249, and 255 of the p53 gene. The mutations result in amino acid substitutions of His Leu at residue 179, Arg Trp at residue 249, and Ile Phe at residue 255 in highly conserved regions of the DNA-binding core domain of the p53 protein. We previously used molecular dynamics calculations to demonstrate that the latter two mutants contain certain common regions that differ substantially in conformation from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of the Leu 179 p53 mutant. The results indicate that the Leu 179 mutant differs substantially from the wild-type structure in certain discrete regions that are similar to those noted previously in the other p53 mutants. One of these regions (residues 204–217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure, but accessible in the mutant structure, and another region (residues 94–110) contains the epitope for the monoclonal antibody PAb1620, which is accessible in the wild-type structure, but concealed in the mutant structure. Immunologic analyses of tumor tissue known to contain this mutation confirmed these predicted conformational shifts in the mutant p53 protein.     

Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: Molecular dynamic and immunologic analyses

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an ArgTrp amino acid substitution at residue 249 and an IlePhe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204–217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.     

Discrimination of single amino acid mutations of the p53 protein by means of deterministic singularities of recurrence quantification analysis

p53 is mutated in roughly 50% of all human tumors, predominantly in the DNA-binding domain codons. Structural, biochemical, and functional studies have reported that the different p53 mutants possess a broad range of behaviors that include the elimination of the tumor-suppression function of wild-type protein, the acquisition of dominant-negative function over the wild-type form, and the establishment of gain-of-function activities. The contribution of each of these types of mutations to tumor progression, grade of malignancy, and response to anticancer treatments has been so far analyzed only for a few "hot-spots." In an attempt to identify new approaches to systematically characterize the complete spectrum of p53 mutations, we applied recurrence quantification analysis (RQA), a non-linear signal analysis technique, to p53 primary structure. Moving from the study of the p53 hydrophobicity pattern, which revealed important similarities with the singular deterministic structuring of prions, we could statistically discriminate, on a pure amino acid sequence basis, between experimentally characterized DNA-contact defective and conformational p53 mutants with a very high percentage of success. This result indicates that RQA is a mathematical tool particularly advantageous for the development of a database of p53 mutations that integrates epidemiological data with structural and functional categorizations.     

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r² = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.     

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

Kinetic instability of p53 core domain mutants: implications for rescue by small molecules

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain. Many of these mutants are thermodynamically unstable at body temperature. Here we show that these mutants also denature within minutes at 37 degrees C. The half-life (t(1/2)) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increasing thermodynamic instability. The highly destabilized mutant I195T had a t(1/2) of less than 1 min. The wild-type p53-(94-360) construct, containing the core and tetramerization domains, was more stable, with t(1/2) = 37 min at 37 degrees C, similar to full-length p53. After unfolding, the denatured proteins aggregated, the rate increasing with higher concentrations of protein. A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain. Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it.     

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.     

Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant

Background

The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298–306 K).

Methodology/Principal Findings

This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately.

Conclusions

The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.     

Binding of p53-derived ligands to MDM2 induces a variety of long range conformational changes

We have used NMR to study the effects of peptide binding on the N-terminal p53-binding domain of human MDM2 (residues 25-109). There were changes in HSQC-chemical shifts throughout the domain on binding four different p53-derived peptide ligands that were significantly large to be indicative of global conformational changes. Large changes in chemical shift were observed in two main regions: the peptide-binding cleft that directly binds the p53 ligands; and the hinge regions connecting the beta-sheet and alpha-helical structures that form the binding cleft. These conformational changes reflect the adaptation of the cleft on binding peptide ligands that differ in length and amino acid composition. Different ligands may induce different conformational transitions in MDM2 that could be responsible for its function. The dynamic nature of MDM2 might be important in the design of anti-cancer drugs that are targeted to its p53-binding site.     

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.     

Structural distortion of p53 by the mutation R249S and its rescue by a designed peptide: implications for "mutant conformation"

Missense mutations in the DNA-binding core domain of the tumour suppressor protein p53 are frequent in cancer. Many of them result in loss of native structure. The mutation R249S is one of the six most common cancer-associated p53 mutations ("hot-spots"). As it is highly frequent in hepatocellular carcinoma, its rescue is an important therapeutic target. We have used NMR techniques to study the structural effects of the R249S mutation. The overall fold of the core domain is retained in R249S, and it does not take up a denatured "mutant conformation". However, the beta-sandwich had increased flexibility and, according to changes in chemical shift, there was local distortion throughout the DNA-binding interface. It is likely that the R249S mutation resulted in an ensemble of native and native-like conformations in a dynamic equilibrium. The peptide FL-CDB3 that was designed to rescue mutants of p53 by binding specifically to its native structure was found to revert the chemical shifts of R249S back towards the wild-type values and so reverse the structural effects of mutation. We discuss the implications for a rescue strategy and also for the analysis of antibody-binding data.     

Gain of function of mutant p53 by coaggregation with multiple tumor suppressors

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.     

p53蛋白质Gly249和Ser249替换型三维结构的分子动力学研究

利用p53蛋白质核心区晶体结构作分子动力学发现,除了生化方面的稳定性之外,该区还具有分子力学上的高度稳定性.在此基础上作的R249残基替换分子动力学研究显示,p53蛋白质核心区249位点精氨酸被其他残基替换后能引起p53蛋白质核心区L2、L3结构域间的密切联系趋于松散,正常的空间构象发生改变并使整个核心区结构稳定性受到破坏.这一研究从三维结构变化上,直观地解释了R249残基替换造成的p53蛋白质免疫和生化特性改变的结构机制.     

Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling.

Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.     

P53蛋白质R175残基替换的分子动力学研究

利用Ｐ５３蛋白核心区晶体结构作分子动力学研究发现,除了生经方面的稳定性之外,该区还具有分子动力学上的高度稳定性。在此基础上作的Ｒ１７５残基替换分子动力学研究显示,Ｐ５３蛋白质核心区１７５位点精氨酸被其他残基替换后能引起Ｐ５３蛋白质核心区Ｌ２、Ｌ３结构域间的密切联系趋于松散,下沉遥空间构象发生改变并使整个核心区结构稳定性受到破坏。这一研究从三维结构变化上,直观地解释了Ｒ１７５钱基替换造成的５３蛋白质