Document Type
Article
Publication Date
2022
Digital Object Identifier (DOI)
https://doi.org/10.3390/biom12111558
Abstract
An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is −3.0 kcal/mol at physiological ionic strength. ΔΔG increased to −1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to −1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure.
Rights Information
This work is licensed under a Creative Commons Attribution 4.0 License.
Was this content written or created while at USF?
Yes
Citation / Publisher Attribution
Biomolecules, v. 12, issue 11, art. 1558
Scholar Commons Citation
Gregory, Emily and Daughdrill, Gary W., "Sequence Properties of an Intramolecular Interaction That Inhibits P53 DNA Binding" (2022). Molecular Biosciences Faculty Publications. 142.
https://digitalcommons.usf.edu/bcm_facpub/142
