Variation at the R181 residue of p53 confers loss of p53 DNA binding cooperativity with the retention of mitochondrial-associated apoptosis

TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. p53 primarily acts as a transcription factor and binds to target sites on DNA cooperatively as a tetramer. This cooperative binding is mediated by salt-bridge interactions between p53 residues E180 and R181 from two different p53 monomers. Variants at the R181 residue are one of the most identified TP53 pathogenic variants by germline genetic testing, however the mechanism by which these variants disrupt p53 tumor suppression is not understood. We show that families with TP53 p.R181H and p.R181C variants have an attenuated cancer risk phenotype compared to patients with hotspot loss of function TP53 variants. Despite this clinical phenotype, we find that p53 R181H and R181C variants have significantly diminished ability to transactivate a set of ~300 known p53 target genes in CRISPR knock-in colorectal and breast cancer cell lines. This loss of transactivation ability does not occur through defects in p53 structure or oligomerization, but through reduced cooperative binding to p53 target sites on DNA as determined using fluorescence polarization assays on purified p53 proteins and using chromatin immunoprecipitation sequencing in R181-mutant cancer cells. Despite the complete loss of p53's transcriptional function, R181 mutants retain some tumor suppressive function. Colony formation assays show efficient colony suppression by R181H and R181C, and injecting R181 knock-in cancer cells into the subcutaneous tissue of mice results in comparable tumor progression levels between R181H, R181C, and wild-type p53. Interestingly, we observe residual apoptotic activity in R181H and R181C mutant cells when treated with DNA-damaging agent 5-fluorouracil, despite the poor transactivation of p53's proapoptotic targets. This suggests that the R181 mutants retain the p53 transcription-independent mechanism of apoptosis, where p53 goes to the mitochondria to induce apoptosis. Indeed, proximity ligation assays between p53 and mitochondrial BAK show that R181 mutants traffic to the mitochondria upon genotoxic stress. Our study elucidates p53 tumor suppressive activities that are lost versus retained by R181 variants, which is estimated to account for 0.5% of all p53 missense mutations.