Formation of Disulfide Bond in p53 Correlates with Inhibition of DNA Binding and Tetramerization

Abstract

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

p53-Induced DNA Bending: The Interplay between p53−DNA and p53−p53 Interactions

“…Biophysical and biochemical data 11,[21][22][23][24] were obtained and a similar p53 core domain tetramer-DNA complex model was proposed 11,[21][22][23][24] . The specific ways of association between p53 and DNA and between p53 monomers are supported by experiments 18,[25][26][27] . In addition, biophysical studies show that the p53 core domain alone can bend the DNA by only 30-35 degrees while the full length p53 can induce bending of 50-70 degrees, depending on the techniques used in the estimation 14 .…”

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

“…The bidirectional regulation of this kinase is well suited for explaining the paradoxical effects of oxidants. In addition to PKC, various specific molecular targets that are directly modified and either inactivated or activated by oxidants have been identified, including protein-tyrosine phosphatase, Ras, and transcriptional factors c-Jun and p53 (55)(56)(57)(58). In contrast, some targets, such as MAPK, are indirectly acti- Neurite outgrowth was measured after 3 days.…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Multiple cysteines in murine p53 were found to be functionally important, and their selective mutation afftected various aspects of its function [202]. S-glutathionylation of cysteine 82 of p53 has been linked to decreased binding to DNA in association with the formation of monomers and high MW oligomers [203]. Interestingly, the redox state of cysteine 277 of human p53 has been linked to its ability to bind to bind p53 response elements in a sequence specific manner [204].…”