title: 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.
authors.affiliation: From the ‡Proteomics Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: Adán
authors.initials: A
authors.lastname: Alpízar
authors.affiliation: §Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands.|¶Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands.
authors.forename: Fabio
authors.initials: F
authors.lastname: Marino
authors.affiliation: ‖Proteobotics SL, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ramos-Fernández
authors.affiliation: From the ‡Proteomics Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Lombardía
authors.affiliation: §Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands.|¶Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands.
authors.forename: Anita
authors.initials: A
authors.lastname: Jeko
authors.affiliation: **Computational Systems Biology Group, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: Florencio
authors.initials: F
authors.lastname: Pazos
authors.affiliation: From the ‡Proteomics Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: Alberto
authors.identifier: 0000-0001-6837-7056
authors.initials: A
authors.lastname: Paradela
authors.affiliation: ‡‡Macromolecular X-ray Crystallography Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain.
authors.forename: César
authors.identifier: 0000-0002-5149-1722
authors.initials: C
authors.lastname: Santiago
authors.affiliation: §Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; A.J.R.Heck@uu.nl mmarcilla@cnb.csic.es.|¶Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, the Netherlands.
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJ
authors.lastname: Heck
authors.affiliation: From the ‡Proteomics Unit, Spanish National Biotechnology Centre (CSIC), Darwin 3, 28049, Madrid, Spain; A.J.R.Heck@uu.nl mmarcilla@cnb.csic.es.
authors.forename: Miguel
authors.identifier: 0000-0001-9171-5076
authors.initials: M
authors.lastname: Marcilla
mesh_terms: D020816:Amino Acid Motifs; D002460:Cell Line; D018360:Crystallography, X-Ray; D015235:HLA-B Antigens / Q000737:chemistry* / Q000378:metabolism*; D059947:HLA-B40 Antigen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*
chemical_list: D015235:HLA-B Antigens; D059947:HLA-B40 Antigen; D010455:Peptides