Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization.
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.
Department of Chemistry and Biochemistry, California State University, Los Angeles, CA 90032, USA.
Xiu Zhu
XZ
Sun
Christopher
C
Vinci
Linna
L
Makmura
Shubo
S
Han
Dung
D
Tran
John
J
Nguyen
Michael
M
Hamann
Sandra
S
Grazziani
Shelether
S
Sheppard
Margarita
M
Gutova
Feimeng
F
Zhou
James
J
Thomas
Jamil
J
Momand
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008970:Molecular Weight; D009579:Nitrobenzoates / Q000737:chemistry; D009687:Nuclear Proteins / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013434:Sulfenic Acids / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013441:Sulfinic Acids / Q000032:analysis; D013451:Sulfonic Acids / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism; D000097642:GADD45 Proteins
D003545:Cysteine; D004247:DNA; D004268:DNA-Binding Proteins; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides; D009579:Nitrobenzoates; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011092:Polyethylene Glycols; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; D013441:Sulfinic Acids; D013451:Sulfonic Acids; D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins; C068748:N-(2-methoxyphenyl)maleimide; C041724:2-nitro-5-thiosulfobenzoic acid; C497206:MDM2 protein, human