Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveals that the Oncogenic Y220C Variant is a Loss of Function Mutation for Zn 2+ -binding at Physiological Temperature

Abstract We present evidence of previously unrecognized allosteric connectivity across the TP53 DNA binding domain (DBD). Specifically, we have found evidence of explicit influence on the Zn 2+ -binding site from the region surrounding the hotspot Y220C mutation. This allosteric connectivity is intertwined with a temperature-dependent destabilization of Zn 2+ binding in both the WT and Y220C DBDs. Our studies indicate that the Y220C mutation exacerbates this temperature-dependent destabilization of Zn 2+ -binding to result in overall destabilization of the Y220C variant. We provide detailed thermodynamic evidence that Rezatapopt, a small molecule reactivator of the Y220C DBD, engages Y220C through two distinct thermodynamic pathways and restores WT-level Zn 2+ -affinity to this oncogenic variant. A series of thermodynamic models describing the WT and Y220C conformational landscapes, as well as the Rezatapopt binding mechanisms, are proposed.