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

• Published in: Molecular & cellular proteomics Vol. 16; no. 2; pp. 181 - 193
• Main Authors: Alpízar, Adán, Marino, Fabio, Ramos-Fernández, Antonio, Lombardía, Manuel, Jeko, Anita, Pazos, Florencio, Paradela, Alberto, Santiago, César, Heck, Albert J.R., Marcilla, Miguel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2017; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology
• Subjects: Aberration; Amino Acid Motifs; Antigen presentation; Antigenic determinants; Antigens; Binding; Cancer; Cancer immunotherapy; Cell Line; Coordination compounds; Crystal structure; Crystallography, X-Ray; HLA-B Antigens - chemistry; HLA-B Antigens - metabolism; HLA-B40 Antigen - chemistry; HLA-B40 Antigen - metabolism; Humans; Immunotherapy; Kinases; Leukocytes; Ligands; Lymphocytes T; Models, Molecular; Molecular structure; Peptides; Peptides - analysis; Peptides - chemistry; Phosphorylation; Post-translation; Protein Binding; Proteomics - methods; Residues; Tumor cells; Workflow
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M116.063800

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.