Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Gary Wayne Daughdrill, Ph.D.

Committee Member

Yu Chen, Ph.D.

Committee Member

Kristina Schmidt, Ph.D.

Committee Member

Libin Ye, Ph.D.

Keywords

Effective concentration, Cooperativity, Fluorescence anisotropy, Intrinsically disordered proteins

Abstract

The intrinsically disordered transcription factor and tumor suppressor p53 binds to promoter response element DNA upon cellular stress and activates genes associated with cell cycle arrest, senescence, and apoptosis. Disruption of sequence specific binding to target gene promoters is heavily implicated in human health, where a majority of cancers contain mutations localized to the DNA binding domain (DBD) of p53. p53 DNA binding is regulated by posttranslational modifications, associations with cellular factors, and by an autoinhibitory intramolecular interaction. The autoinhibitory intramolecular interaction occurs when the disordered N-terminal transactivation domain (TAD) interacts with the ordered DBD. Previous work in the Daughdrill lab showed that the second transactivation domain (TAD2) and the proline rich region (PRR) are responsible for inhibition of DNA binding. The goal of this study is to investigate the specific features of TAD2 and PRR that result in inhibition and to gain insight into how these interactions regulate DNA binding.

The interaction of the disordered TAD2 and PRR with DBD is multivalent and dynamic. We studied fragments of p53 that included only the DBD and a minimal fragment with maximal inhibition of DNA binding that includes TAD2, PRR and the DBD (ND). We then systematically mutated physicochemical features in TAD2 and PRR to reduce or eliminate inhibition of DNA binding. The TAD2 mutants targeted the negatively charged residues of TAD2, nonpolar residues of TAD2, a conserved motif implicated in p53 transactivation, or a complete deletion of TAD2. PRR mutants were designed to eliminate chain stiffness due to proline content, potential nonpolar interactions between PRR and DBD, a known pi-cation interaction between PRR residue W91 and DBD residue R174, or to replace the PRR with a flexible linker composed of alternating Gly, Ser, and Thr residues. The effects of these mutations on DNA binding affinity to target and nontarget DNA sequences were measured using fluorescence anisotropy and analytical size exclusion chromatography was used to measure changes in the Stokes radius of p53 ND.

We find TAD2 mutations moderately restore DNA binding to ND, disrupting the intramolecular interaction and increasing the Stokes radius. By analyzing DNA binding under varying salt concentrations using the counterion condensation theory, we find a change in the apparent excess ion release mediated by the charged residues of TAD2, suggesting a mechanism of energetic control over the DNA binding process. We find the PRR is directly involved in autoinhibition but also has a frustrating effect on the interaction between TAD2 and DBD. When TAD2 is deleted, PRR can inhibit DNA binding by a factor of 10 compared to DBD but when TAD2 is present PRR controls its orientation and reduces its ability to inhibit DNA binding.

Analysis of the effective concentration of TAD2 based on PRR suggests autoinhibition is not optimized. Evolutionary analysis suggests the intramolecular interaction is likely present in birds and most mammals, and the frustrated component may have emerged simultaneously.

The results of our experiments define a system where the molecular features of TAD2 and PRR simultaneously compete and cooperate to maintain optimal autoinhibition.

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