摘要:SummaryPhosphopeptides presented by major histocompatibility complex (MHC) class I have been regarded as a pivotal type of cancer neoantigens that are recognized by T cells. The structural basis of single-phosphorylated peptide presentation has been well studied. Diphosphorylation with one interval between two sites is one of the prevalent forms of multisite-phosphorylated peptides. Herein, we determined the molecular basis of presentation of two P4/P6 double pS-containing peptides by HLA-B27 and compared them with unmodified and single-phosphorylated peptide complexes. These data clarified not only the HLA allele-specific presentation of phosphopeptides by MHC class I molecules but also the cooperativity of peptide conformation within P4 and P6 phosphorylation sites. The phosphorylation of P6 site can influence the binding mode of P4 phosphorylated site to HLA-B27. And we found the diphospho-dependent attenuated effect of peptide binding affinity. This study provides insights into the MHC presentation features of diphosphopeptides, which is different from monophosphopeptides.Graphical abstractDisplay OmittedHighlights•Diphosphorylation with an interval is prevalent among multiphosphorylated peptides•Diphosphopeptide presentation is HLA specific and different from monophosphopeptide•The cooperativity of peptide conformation within P4 and P6 phosphorylation sites•The diphosphorylation attenuates peptide binding affinity to HLA-B27Biomolecules; Peptides; Immunology; Crystallography