Phosphorylation of a Tumor-Derived ASXL2 Epitope Remodels the HLA-Bound Peptide Conformational Ensemble and Interaction Network of the Peptide-HLA Complex.

The expansion of cancer immunotherapy has made it increasingly important to identify tumor-specific human leukocyte antigen (HLA) class I epitopes and to define the molecular determinants of peptide-HLA binding and stability. Posttranslationally modified epitopes, particularly phosphorylation, can reflect oncogenic signaling states and offer highly tumor-selective targets. Despite their potential, the structural and biophysical mechanisms by which phosphorylation reshapes the conformational ensemble and interaction network of peptide-HLA complexes remain unclear. Here, we investigate a phosphorylated ASXL transcriptional regulator 2 (ASXL2)-derived epitope supported by confident tandem mass spectrometry-based phosphosite localization and detected across multiple cancer types. We performed all-atom molecular dynamics simulations of peptide-HLA complexes to compare the phosphorylated peptide, its nonphosphorylated counterpart and its protonated-phosphorylated state. Our simulations indicate that phosphorylation reconfigures the noncovalent interaction network within the binding groove by introducing new local contacts. Principal component analysis further revealed that phosphorylation restricts peptide conformational fluctuations, thereby altering the conformational ensemble of the peptide-HLA complex. Furthermore, protonation of the phosphorylated peptide remodels the noncovalent interaction network and conformational ensemble. Together, these findings provide a structural and biophysical basis for how phosphorylation can modulate the conformational ensemble and interaction network of peptide-HLA complexes, which may help prioritize cancer-specific targets for immunotherapeutic development.