Phosphorylated self-peptides alter human leukocyte antigen class I-restricted antigen presentation and generate tumor-specific epitopes

Abstract

Human leukocyte antigen (HLA) class I molecules present a variety of posttranslationally modified epitopes at the cell surface, although the consequences of such presentation remain largely unclear. Phosphorylation plays a critical cellular role, and deregulation in phosphate metabolism is associated with disease, including autoimmunity and tumor immunity. We have solved the high-resolution structures of 3 HLA A2-restricted phosphopeptides associated with tumor immunity and compared them with the structures of their nonphosphorylated counterparts. Phosphorylation of the epitope was observed to affect the structure and mobility of the bound epitope. In addition, the phosphoamino acid stabilized the HLA peptide complex in an epitope-specific manner and was observed to exhibit discrete flexibility within the antigen-binding cleft. Collectively, our data suggest that phosphorylation generates neoepitopes that represent demanding targets for T-cell receptor ligation. These findings provide insights into the mode of phosphopeptide presentation by HLA as well as providing a platform for the rational design of a generation of posttranslationally modified tumor vaccines.

Fig. 1.

Peptide conformations within the antigen-binding cleft. CDC25b (A), IRS2 (B), β-catenin (C), CDC25b-phospho (D), IRS2-phospho (E), and β-catenin–phospho (F). Blue mesh indicates unbiased 2Fo-Fc maps contoured at 1σ. Yellow indicates nonphosphorylated peptides. Green indicates phosphorylated peptides. The bound peptide is shown from a side-on view with the α2-helix removed for clarity.

Fig. 2.

Interactions of the phosphorylation site in nonphosphopeptide and phosphopeptide HLA A2 complexes. Accommodation of the phosphate moiety by HLA A2 is accompanied by changed interactions in the complex, adding to the differential presentation of altered self. Stick representation of peptides and of heavy-chain side chains that interact with the phosphorylation site. Yellow indicates nonphospho-pHLA A2 complexes. Green indicates phospho-pHLA A2 complexes. (A–C) Phosphorylation of P5-Ser in CDC25b leads to an altered peptide conformation attributable to steric constraints. (D–F) Phosphorylation of P4-Ser in IRS2 gives rise to numerous interactions and subtly alters the conformation of Arg 65 and Lys 66. (G–I) Phosphorylation of P4-Ser in β-catenin stabilizes the mobile peptide residues P3 to P6.

Fig. 3.

Altered surface potential for TCR recognition. Surface representation of the HLA A2 with bound peptides. CDC25b (A), IRS2 (B), CDC25b-phospho (C), and IRS2-phospho (D). Gray indicates α-chain, with putative TCR contact residues in purple, based on the structure of the A6/HLA A2-Tax complex structure (25). The arrows indicate the peptide phosphorylation sites. The negatively charged phosphate groups are located within the area of a typical TCR footprint and are likely to dominate TCR discrimination. Electrostatic potentials (blue, positive; red, negative) were calculated with APBS (22). The phosphoserine residues were assumed to carry 2 negative charges.

Fig. 4.

Stabilization experiments with the peptides GLLGpSPVRA, RVApSPTSGV, and YLDpSGIHSGA and their native forms. (A, D, and G) Thermal denaturation of the phosphopeptide- and nonphosphopeptide-MHC complexes using CD spectroscopy. (B, E, and H) Competition-based peptide binding assay with the phosphopeptides and nonphosphopeptides. (C, F, and I) Dephosphorylation of the single phosphopeptides and the phosphopeptide-MHC complexes by alkaline phosphatase. Nonphosphorylated peptide/complexes are shown by gray lines and phosphopeptide/complexes are shown by black lines.