Oxidation of p53 through DNA Charge Transport Involves a Network of Disulfides within the DNA-Binding Domain
Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were ^(13)C_2D_2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward ^(13)C_2D_2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.
© 2015 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: November 17, 2014; Revised: December 19, 2014; Publication Date (Web): January 13, 2015. This research was funded by the Ellison Foundation (AG-SS-2079-08), the Moore Foundation for support of the Center for Chemical Signaling at Caltech, the Gordon and Betty Moore Foundation (GBMF775), The Beckman Institute, and National Institutes of Health (1S10OD010788-01A1). We thank Lisa Beckmann for assistance with molecular cloning and protein purification.
Published - bi501424v.pdf
Supplemental Material - bi501424v_si_001.pdf