A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens

Abstract

As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.

Fig. 1.

Sequence analysis of the phosphorylated and the nonphosphorylated peptides associated to HLA-B*40. Only the data corresponding to peptides of 9 to 11 residues long are shown. A, Frequency distribution of phosphorylation among the identified HLA-B*40 phospholigands. B and C, Sequence logos of the phosphopeptides (B) and the nonmodified peptides (C) identified in the HLA-B*40 ligandome.

Fig. 2.

Frequency distribution of phosphorylation among the identified HLA-B*40 phosphopeptides (blue) and the in silico predicted HLA-B*40 binders (red).

Fig. 3.

Peptide binding assays to HLA-B*40. Bars show the mean ± S.D. of three independent experiments. Statistically significant differences are indicated (** p < 0.01; * = p < 0.05). A, Binding affinities -expressed as IC₅₀ values- of seven phospholigands associated to HLA-B*40 and their nonphosphorylated counterparts. In all cases, the pair of peptides exhibit alike affinities although the affinity of the phosphopeptides is somewhat lower. B, Binding affinities of 12 phosphorylated poly-Gly analogs with Gly (blue) or Arg (red) at P1.

Fig. 4.

Crystal structures of HLA-B*40 in complex with the peptides REF(pS)KEPEL or REFSKEPEL. A and B, Structure and electron density map (2Fo-Fc) at 1σ (blue mesh) of the peptides REFSKEPEL (A), and REF(pS)KEPEL (B) anchored to the B*40 binding cleft. C, Superposition of the binding grooves of both complexes with the peptides REFSKEPEL (green) and REF(pS)KEPEL (yellow) displayed in ribbon style.

Fig. 5.

Overview of the interactions of residues at P1 and P4 of the peptides REF(pS)KEPEL (A and B) and REFSKEPEL C and D, with the binding groove of HLA-B*40. (A) Interactions involving residues P1-Arg and P4-pSer in the complex B*40-REF(pS)KEPEL. The carbon atoms of the peptide ligand are depicted in yellow. B, Schematic representation of the interactions displayed in panel A. C, Interactions involving residues P1-Arg and P4-pSer in the complex B*40-REFSKEPEL. The carbon atoms of the peptide ligand are depicted in green. D, Schematic representation of the interactions displayed in panel C.

Fig. 6.

Sequence analysis of the phosphorylated and the nonphosphorylated peptides associated to HLA-B*39 (left column), HLA-B*27 (middle column) or HLA-B*07 (right column). For clarity, only the data corresponding to 9-mer peptides are shown. For other peptide lengths, see supplemental Data S8. A, Frequency distribution of phosphorylation at each peptide position in the phospholigandomes bound to these allotypes, revealing the preference for phosphorylation at P4. B and C, Sequence logos of the phosphopeptides (B) and the nonmodified peptides (C) identified in the ligandomes of these HLA-B molecules.