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

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.