Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Gary Wayne Daughdrill, Ph.D.
Younghoon Kee, Ph.D.
Meera Nanjundan, Ph.D.
Sameer Varma, Ph.D.
Intrinsically disordered proteins, NMR, phosphomimetics, phosphorylation, transient secondary structure
p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome degradation. In turn, p53 activates the transcription of Mdm2. This negative feedback loop not only regulates p53 levels but also the fate of the cell. In stressful conditions, such as DNA damage, p53 levels increase within the nucleus, where it becomes active and induces cell growth arrest or apoptosis.
p53 consists of discrete domains that participate in sequence-specific DNA binding, tetramerization, and transcriptional activation. p53 contains two transactivation domains (TAD1 and TAD2), that contain multiple phosphorylation sites. The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, Mdm2 and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetic mutations on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3-5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2/MdmX interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with p53 negative regulators.
Previous studies have shown that there is an intramolecular interaction between p53TAD and the DNA binding domain of p53 and that this interaction can reduce sequence-specific DNA binding. We aim to determine whether mutations within TAD2 combined with site-specific phosphorylation can decrease p53-DNA binding. Two consecutive hydrophobic residues within TAD2 (W53, F54), which are surrounded by acidic amino acids, are essential for the activity of the transactivation domain. We will determine the effect that these mutated sites have on DNA binding. Using biophysical methods, we investigated the effects of point mutations and phosphomimetic mutations on the interaction between p53 and DNA. Our binding data shows that phosphomimicry of the p53TAD decreases DNA binding affinity, while substituting the hydrophobic residues, (W53, F54), with acidic residues within TAD2 of p53 increases DNA binding. By determining the impact that phosphorylation has on the binding affinity of p53 we will identify a new understanding of the structure and function of not just p53 but other intrinsically disordered proteins.
Scholar Commons Citation
Levy, Robin, "Posttranslational Modification and Protein Disorder Regulate Protein-Protein Interactions and DNA Binding Specificity of p53" (2020). USF Tampa Graduate Theses and Dissertations.