Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveal that the Oncogenic Y220C Variant is a Loss-of-Function Mutation for Zn2+-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 an explicit influence on the Zn2+-binding site from the region surrounding the hotspot Y220C mutation. This allosteric connectivity is intertwined with a temperature-dependent destabilization of Zn2+ binding in both the WT and the Y220C DBDs. Our studies indicate that the Y220C mutation exacerbates this temperature-dependent destabilization of Zn2+ 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 thermodynamically distinct binding mechanisms and restores WT-level Zn2+-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.