Graduation Year
2024
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
Kristina Schmidt, Ph.D.
Committee Member
Jiandong Chen, Ph.D.
Committee Member
Libin Ye, Ph.D.
Keywords
Intrinsically disordered proteins, isothermal titration calorimetry, molecular dynamics simulation, nuclear magnetic resonance
Abstract
p53 is an intrinsically disordered protein that is a tumor suppressor. The transactivation domain is split into two parts (TAD1 and TAD2). These TADs bind to inhibitors MDM2, and a homolog, MDMX, but TAD1 binds with greater affinity. MDM2 is an E3 ubiquitin ligase that leads to the degradation of p53. MDMX is not an E3 ubiquitin ligase, but it does prevent p53 from binding to DNA, thus preventing its tumor suppressor role.
Previous research showed that increasing p53 helicity by mutating prolines to alanines increased p53's helicity but not the length of the helix. The increase in helicity increased the binding affinity to MDM2. It also showed that there were differences in the changes of chemical shifts of MDM2 residues outside of the primary binding pocket between binding to wild type p53 and a proline-27-to-alanine mutant, it was hypothesized that the increase in binding was due to a change in conformational entropy.
Presented is a comprehensive thermodynamic analysis of the p53 tumor suppressor binding to MDM2 and MDMX. Isothermal titration calorimetry (ITC) was used to measure ΔH, ΔS, and ΔCp for three different fragments of the p53 transactivation domain, containing residues 16-29, 17-35, and 1-73 binding to MDM2 and MDMX. We also performed ITC with a helical p53 mutant P27A. For all the p53 variants, binding to both MDM2 and MDMX is dominated by the hydrophobic effect as evidenced by negative ΔCp values. These values have a characteristic shift from a more entropically driven process at lower temperatures to a more enthalpically driven process at higher temperatures. KD values for p5316-29 binding to MDM2 and MDMX were consistent with earlier reports. However, when residues 30-35 were added to p53, the KD for binding MDM2 decreased by 2-fold while the KD for binding MDMX decreased by 10-fold. The higher binding affinity to MDMX and more negative ΔCp than MDM2 for p5317-35 is due to the stabilization of MDMX and no additional interactions with a secondary binding pocket. KD values for the more helical P27A mutant were also consistent with earlier reports and a comparison of the temperature dependence of ΔH and -TΔS clearly shows that wild type p53 has a larger entropic penalty for coupled folding and binding than P27A. ΔCp measurements of p5316-29 and p5317-35 binding to MDM2 fragments with and without the N- and C-terminal disordered regions showed that access to the secondary binding pocket is inhibited by the C-terminal disordered region. Interestingly, the C-terminal disordered region of MDM2 did not interfere with access to the secondary binding pocket for a more helical mutant of p53 that substitutes proline 27 for alanine. This result is supported by our all-atom molecular dynamics (MD) simulations showing that p53 residues 31-35 turn away from the C-terminal disordered region of MDM2 in P27A17-35 and make direct contact with this region in p5317-35. We propose that the C-terminal tail is displaced when wild type p53 binds the secondary site but is not displaced when the mutant binds. In addition, the probability contact maps from the MD simulations of p5317-35 binding to either MDM2 or MDMX are consistent with chemical shift changes from the nuclear magnetic resonance (NMR) titrations, providing additional evidence for a secondary binding pocket. The MD simulations also show that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site.
ΔASA values from the MD simulations are similar for p5317-35 bound to either MDM2 or MDMX. A comparison of the simulation trajectories suggests an energetic contribution other than surface area burial that may be responsible for the tighter binding of p5317-35 to MDMX. The temperature dependence of -TΔS clearly shows that wild type p5316-29 has a larger entropic penalty for coupled folding and binding than P27A16-29, and -TΔS values from ITC are consistent with -TΔS estimates using generalized order parameters from NMR relaxation data. Our results indicate that the entropic penalty of coupled folding and binding for a small intrinsically disordered region like p53TAD can be measured directly with ITC and estimated from generalized order parameters. A better understanding of secondary binding pockets on MDM2 and MDMX will facilitate the design of more potent p53 inhibitors.
Scholar Commons Citation
Higbee, Pirada Serena, "The Thermodynamics and Structure of Tumor Suppressor p53 and Helical Mutants Binding to Inhibitors MDM2 and MDMX at the Primary and Secondary Sites" (2024). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/10809
