Abstract
The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (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 phosphomimetics 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 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 its negative regulators.
Highlights
Loss of function mutations in the p53 pathway frequently arise during cancer development [1,2,3].Approximately half of all human tumors express p53 mutants with reduced DNA-binding affinity which reduces or eliminates transactivation [3,4]
The results argue that binding affinity between phosphorylated and unphosphorylated p53 to mouse double minute 2 (Mdm2)/MdmX is primarily controlled by electrostatics
Using phosphomimetic mutations we investigated the effect of phosphorylation on the binding of p53 with Mdm2, MdmX, and CREB binding protein (CBP)/KIX and the transient secondary structure of p53 transactivation domain (p53TAD)
Summary
Loss of function mutations in the p53 pathway frequently arise during cancer development [1,2,3].Approximately half of all human tumors express p53 mutants with reduced DNA-binding affinity which reduces or eliminates transactivation [3,4]. The tumor suppressor protein p53 is a well-known intrinsically disordered protein (IDP) whose disorder is a major component of its functionality [5,6]. An IDP is a protein that lacks a fixed or ordered structure. IDPs are structurally very different from ordered proteins and tend to have distinct properties in terms of function, sequence, interactions, evolution and regulation [7,8,9]. Many IDPs like p53 form transient secondary structures and undergo coupled folding and binding when they are bound to their targets [10,11,12]. Disordered proteins cover a range of different states from fully unstructured to partially structured [8,13,14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
