Stabilization of mutant p53 via alkylation of cysteines and effects on DNA binding

Abstract

Oncogenic mutations inactivate the tumor suppressor p53 by lowering its stability or by weakening its binding to DNA. Alkylating agents that reactivate mutant p53 are currently being explored for cancer therapy. We have discovered ligands containing an a,b-unsaturated double bond, characteristic of Michael acceptors, that bind covalently to generic cysteine sites in the p53 core domain. They raised the melting temperature of the core domain of wild-type p53 and the hotspot mutants R175H, Y220C, G245S, R249S, and R282 by up to 3°C. Analysis of the relative reactivity of the cysteines in p53 by mass spectrometry found that C124 and C141 react first, followed by C135, C182, and C277, and eventually C176 and C275. Post-translational modifications of cysteines are known to be involved in regulation of other transcription factors. Modification of C277, which sits on the DNA-binding surface, may, for example, play a role in regulating p53 activity in cells in response to environmental cues. We found that the modifications progressively reduced DNA-binding activity of full-length p53. In light of these results, it is likely that the anticancer activity of the alkylating drugs works via a nontranscriptional activity of p53.

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

“…6, that Cys182 and Cys277 are the most accessible cysteine residues on the protein surface. During the preparation of this manuscript, X-ray crystallography data was reported by Kaar et al [44], which is in agreement with the p53 FTICR mass spectrometry results presented here. Stabilised p53 core domain (T-p53C; an engineered p53 variant that contains the four mutations M133L/V203A/N239Y/N268D and displays increased thermostability compared with the wt protein [45]) containing the cancer-derived mutation Y220C was treated with an electrophilic compound, 3-benzoylacrylic acid, and the resulting crystal structure revealed pronounced electron density surrounding cysteine residues 182, 229, and 277.…”

“…Therefore, investigating the order of Cys reactivity using a bottom-up approach requires that an individual isoform of modified protein is completely pure prior to proteolytic digestion. It may be that tryptic peptides originating from p53, which was alkylated beyond Cys182, 229, and 277, were present in the peptide samples analysed by Kaar et al [44] and Velu et al [46], and resulted in the misassignment of Cys reactivity.…”

“…Deconvoluted neutral mass spectra are shown modifications unambiguously. Kaar et al [44] went on to use a bottom-up MS approach to investigate the order of cysteine reactivity in T-p53C-Y220C with 3-benzoylacrylic acid. They reported that they were able to map each Cys residue using MALDI-TOF MS and LC-MS/MS.…”

“…Top-down FTICR MS of 3NEM-p53 revealed that Cys229 and Cys275 are the most likely candidates for the third site of alkylation. Interestingly, X-ray crystallography data has previously indicated that Cys229 is modified by the electrophilic compound 3-benzoylacrylic acid [44]. Both Cys229 and Cys275 are located at the N-terminal and C-terminal ends, respectively, of strands of the anti-parallel β-sheet sandwich.…”

Multisite aggregation of p53 and implications for drug rescue

“…We initially tackled the rescue by stabilizing the folded conformation against unfolding, first by a generic approach using a peptide that binds to native p53 (34) and mutants (35) and then by targeting a specific mutant where the mutation forms a druggable cavity, Y220C. Its melting temperature is raised, and its aggregation is slowed in vitro by small mutant-specific molecules that bind in the cavity (36,37) and rescue Y220C in cancer cell lines (25,38,39). Eisenberg and coworkers (40) have taken a different approach of inhibiting the aggregation process, per se, with a peptide that caps the specific exposed aggregation-prone sequence, centered on Ile254, rather than stabilizing the native structure.…”