Determinants of C₁₆-Ceramide Binding to p53

The tumor suppressor p53 coordinates cellular stress responses, but underlying mechanisms remain incompletely understood. We previously demonstrated that, in response to metabolic stress, C₁₆-ceramide produced by ceramide synthase 6 (CerS6) directly binds to p53, thus preventing its MDM2-mediated degradation and promoting p53 activation. Here, we investigated the structural requirements and functional consequences of ceramide binding to p53. Using a panel of p53 mutants, including naturally occurring oncogenic variants, we characterized the ceramide-binding interface of p53 and the role of amino acid substitutions within this region in metabolic stress signaling. We found that disruption of ceramide binding impaired stress-induced p53-CerS6 interaction at the endoplasmic reticulum (ER), attenuated induction of p53 target genes, and reduced cellular sensitivity to stress. Of note, certain cancer-associated p53 mutants retained ceramide binding and the ability to activate stress responses. These studies were further extended to monitoring the p53-CerS6 interaction on the ER and associated membrane aggregation using fluorescence techniques. We showed that metabolic stress-induced ER remodeling was distinct from the canonical UPR. Overall, our study defines the ceramide-binding surface within the p53 DNA-binding domain and provides novel insight into the functional role of the ceramide-p53 interaction.