Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveals that the Oncogenic Y220C Variant is a Loss of Function Mutation for Zn2+-binding at Physiological Temperature

Stuckey, J. I.; Vivat, V.; Dickson, B. M.; Setser, J. W.; Li, Y.; Cremers, C. M.; Wilson, J. E.; Peterle, D.; Engen, J. R.; Wales, T. E.; Wilcken, R.; Rohan, E. B.; Chenail, G.; Audia, J. E.; Sims, R. J.

doi:10.64898/2026.01.31.701171

bioRxiv · 10.64898/2026.01.31.701171

Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveals that the Oncogenic Y220C Variant is a Loss of Function Mutation for Zn2+-binding at Physiological Temperature

Stuckey, J. I.·Vivat, V.·Dickson, B. M.·Setser, J. W.·Li, Y.·Cremers, C. M.·Wilson, J. E.·Peterle, D.·Engen, J. R.·Wales, T. E.·Wilcken, R.·Rohan, E. B.·Chenail, G.·Audia, J. E.·Sims, R. J.

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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 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 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 distinct thermodynamic pathways 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.

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Stuckey, J. I., Vivat, V., Dickson, B. M., Setser, J. W., Li, Y., Cremers, C. M., Wilson, J. E., Peterle, D., Engen, J. R., Wales, T. E., Wilcken, R., Rohan, E. B., Chenail, G., Audia, J. E., Sims, R. J.. 2026-02-03. Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveals that the Oncogenic Y220C Variant is a Loss of Function Mutation for Zn2+-binding at Physiological Temperature. https://doi.org/10.64898/2026.01.31.701171

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