Phosphorylation of a tumor-derived ASXL2 epitope remodels 1 peptide-HLA binding affinity and interaction dynamics

The expansion of cancer immunotherapy has made it increasingly important to identify tumor-specific HLA class I epitopes and to define the molecular determinants of peptide-HLA binding and stability. Post-translationally modified epitopes, particularly phosphorylation, can reflect oncogenic signaling states and offer highly tumor-selective targets. Despite their potential, the structural and dynamic mechanisms by which phosphorylation reshapes peptide-HLA affinity and stability remain unclear. Here, we investigate a phosphorylated ASXL2-derived epitope supported by confident MS/MS-based phosphosite localization and detected across multiple cancer types. We performed all-atom molecular dynamics simulations of peptide-HLA (pHLA) complexes to compare the phosphorylated peptide, its non-phosphorylated counterpart, and related variants. Our simulations indicate that phosphorylation reconfigures non-bonded interaction networks within the binding groove by introducing new local contacts, leading to an enhanced predicted binding affinity. Furthermore, principal component analysis revealed that phosphorylation increases the overall conformational flexibility and alters the collective backbone motions of the pHLA complex. Together, these findings provide a structural-dynamic basis for how phosphorylation can modulate pHLA stability and interaction dynamics, guiding the rational prioritization and targeting of cancer-specific pHLA complexes for immunotherapy.