title: Impact of the K24N mutation on the transactivation domain of p53 and its binding to murine double-minute clone 2.
abstract: The level of the p53 transcription factor is negatively regulated by the E3 ubiquitin ligase murine double-minute clone 2 (MDM2). The interaction between p53 and MDM2 is essential for the maintenance of genomic integrity for most eukaryotes. Previous structural studies revealed that MDM2 binds to p53 transactivation domain (p53TAD) from residues 17 to 29. The K24N mutation of p53TAD changes a lysine at position 24 to an asparagine. This mutation occurs naturally in the bovine family and is also found in a rare form of human gestational cancer called choriocarcinoma. In this study, we have investigated how the K24N mutation affects the affinity, structure, and dynamics of p53TAD binding to MDM2. Nuclear magnetic resonance studies of p53TAD show that the K24N mutant is more flexible and has less transient helical secondary structure than the wild type. Isothermal titration calorimetry measurements demonstrate that these changes in structure and dynamics do not significantly change the binding affinity for p53TAD-MDM2. Finally, free-energy perturbation and standard molecular dynamic simulations suggest the negligible affinity change is due to a compensating interaction energy between the K24N mutant and the MDM2 when it is bound. Overall, the data suggest that the K24N-MDM2 complex is able to, at least partly, compensate for an increase in the conformational entropy in unbound K24N with an increase in the bound-state electrostatic interaction energy.
authors.affiliation: Department of Physics, University of Idaho, Moscow, Idaho.
authors.forename: Yingqian Ada
authors.initials: YA
authors.lastname: Zhan
authors.forename: Hongwei
authors.initials: H
authors.lastname: Wu
authors.forename: Anne T
authors.initials: AT
authors.lastname: Powell
authors.forename: Gary W
authors.initials: GW
authors.lastname: Daughdrill
authors.forename: F Marty
authors.initials: FM
authors.lastname: Ytreberg
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2
keywords: binding mechanism; intrinsically disordered protein; isothermal titration calorimetry; molecular dynamics; nuclear magnetic resonance spectroscopy