Evolutionary Conservation and Functional Constraint of TP53 Mutation Hotspots Across Mammalian Species

The tumour suppressor protein TP53 is essential for preserving genomic integrity and is among the most frequently mutated genes in human cancers. Mutations in TP53 show a non-uniform distribution and are concentrated at specific residues, commonly referred to as mutation hotspots. Although these positions are well characterised in clinical studies, their evolutionary behaviour across species remains insufficiently understood. In this study, TP53 protein sequences from ten mammalian species were analysed to investigate conservation patterns at six canonical hotspot residues (R175, G245, R248, R249, R273, and R282). Multiple sequence alignment was performed, followed by residue-level conservation scoring and statistical comparison with non-hotspot positions. Hotspot residues exhibited significantly higher conservation (mean ≈ 0.95) compared to non-hotspot residues (mean ≈ 0.78), with the difference confirmed by the Mann–Whitney U test (p = 0.034). In addition, hotspot positions showed reduced variability, indicating stronger evolutionary constraint. Sequence identity analysis revealed moderate to high similarity (69–92%) across species, supporting the robustness of the dataset. Structural and alignment-based observations further demonstrated that hotspot residues are localised within a highly conserved functional domain. These findings suggest that TP53 mutation hotspots represent evolutionarily constrained residues embedded within critical structural regions, providing insight into their susceptibility to recurrent mutation in cancer.