Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self

Nature Immunology volume 9, pages 1236–1243 (2008)Cite this article

Abstract

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of 'transformed self'. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide–HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Bioinformatics characterization of the HLA-A2–restricted phosphopeptide repertoire.
Figure 2: Overall structure of the RTY–HLA-A2 complex.
Figure 3: Interactions of the P4 p-Ser with the HLA-A2 heavy chain.
Figure 4: Accommodation of subdominant anchor residues in phosphopeptide–HLA-A2 complexes.
Figure 5: Interaction of the solvent-exposed phosphate moiety with a positively charged region of the peptide-MHC surface.
Figure 6: Potential influence of the phosphate moiety on antigenic identity.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Rock, K.L. & Goldberg, A.L. Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17, 739–779 (1999).

    Article  CAS  Google Scholar 

  2. Engelhard, V.H., Altrich-Vanlith, M., Ostankovitch, M. & Zarling, A.L. Post-translational modifications of naturally processed MHC-binding epitopes. Curr. Opin. Immunol. 18, 92–97 (2006).

    Article  CAS  Google Scholar 

  3. Zarling, A.L. et al. Phosphorylated peptides are naturally processed and presented by major histocompatibility complex class I molecules in vivo. J. Exp. Med. 192, 1755–1762 (2000).

    Article  CAS  Google Scholar 

  4. Zarling, A.L. et al. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy. Proc. Natl. Acad. Sci. USA 103, 14889–14894 (2006).

    Article  CAS  Google Scholar 

  5. Andersen, M.H. et al. Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL. J. Immunol. 163, 3812–3818 (1999).

    CAS  PubMed  Google Scholar 

  6. Harper, J.W. A phosphorylation-driven ubiquitination switch for cell-cycle control. Trends Cell Biol. 12, 104–107 (2002).

    Article  CAS  Google Scholar 

  7. Koepp, D.M., Harper, J.W. & Elledge, S.J. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97, 431–434 (1999).

    Article  CAS  Google Scholar 

  8. Reed, S.I. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. Nat. Rev. Mol. Cell Biol. 4, 855–864 (2003).

    Article  CAS  Google Scholar 

  9. Ang, X.L. & Wade Harper, J. SCF-mediated protein degradation and cell cycle control. Oncogene 24, 2860–2870 (2005).

    Article  CAS  Google Scholar 

  10. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  11. Wilkinson, K.D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin. Cell Dev. Biol. 11, 141–148 (2000).

    Article  CAS  Google Scholar 

  12. Princiotta, M.F. et al. Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18, 343–354 (2003).

    Article  CAS  Google Scholar 

  13. Blume-Jensen, P. & Hunter, T. Oncogenic kinase signalling. Nature 411, 355–365 (2001).

    Article  CAS  Google Scholar 

  14. Evan, G.I. & Vousden, K.H. Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342–348 (2001).

    Article  CAS  Google Scholar 

  15. Greenman, C. et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).

    Article  CAS  Google Scholar 

  16. Fecher, L.A., Cummings, S.D., Keefe, M.J. & Alani, R.M. Toward a molecular classification of melanoma. J. Clin. Oncol. 25, 1606–1620 (2007).

    Article  CAS  Google Scholar 

  17. Easty, D.J. & Bennett, D.C. Protein tyrosine kinases in malignant melanoma. Melanoma Res. 10, 401–411 (2000).

    Article  CAS  Google Scholar 

  18. Bantscheff, M. et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25, 1035–1044 (2007).

    Article  CAS  Google Scholar 

  19. Diella, F. et al. Phospho.ELM: a database of experimentally verified phosphorylation sites in eukaryotic proteins. BMC Bioinformatics 5, 79 (2004).

    Article  Google Scholar 

  20. Hornbeck, P.V., Chabra, I., Kornhauser, J.M., Skrzypek, E. & Zhang, B. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics 4, 1551–1561 (2004).

    Article  CAS  Google Scholar 

  21. Bjorkman, P.J. et al. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329, 506–512 (1987).

    Article  CAS  Google Scholar 

  22. Madden, D.R., Garboczi, D.N. & Wiley, D.C. The antigenic identity of peptide-MHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2. Cell 75, 693–708 (1993).

    Article  CAS  Google Scholar 

  23. Madden, D.R. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13, 587–622 (1995).

    Article  CAS  Google Scholar 

  24. Ruppert, J. et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74, 929–937 (1993).

    Article  CAS  Google Scholar 

  25. Bouvier, M. & Wiley, D.C. Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules. Science 265, 398–402 (1994).

    Article  CAS  Google Scholar 

  26. Fersht, A.R. et al. Hydrogen bonding and biological specificity analysed by protein engineering. Nature 314, 235–238 (1985).

    Article  CAS  Google Scholar 

  27. Sette, A. et al. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol. 153, 5586–5592 (1994).

    CAS  PubMed  Google Scholar 

  28. Hendrickson, R.C., Skipper, J.C., Shabanowitz, J., Slingluff, C.L., Jr. & Engelhard, V.H . in Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (ed. Lefkovits, I.) 605–623 (Academic, New York, 1996).

    Google Scholar 

  29. Martin, S.E., Shabanowitz, J., Hunt, D.F. & Marto, J.A. Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 72, 4266–4274 (2000).

    Article  CAS  Google Scholar 

  30. Sidney, J. et al. Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules. Hum. Immunol. 62, 1200–1216 (2001).

    Article  CAS  Google Scholar 

  31. Gulukota, K., Sidney, J., Sette, A. & DeLisi, C. Two complementary methods for predicting peptides binding major histocompatibility complex molecules. J. Mol. Biol. 267, 1258–1267 (1997).

    Article  CAS  Google Scholar 

  32. Garboczi, D.N., Hung, D.T. & Wiley, D.C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89, 3429–3433 (1992).

    Article  CAS  Google Scholar 

  33. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  34. Collaborative Computational Project No 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  35. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  36. Brünger, A.T. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992).

    Article  Google Scholar 

  37. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  38. Fenn, T.D., Ringe, D. & Petsko, G.A. POVScript+: a program for model and data visualization using persistence of vision ray-tracing. J. Appl. Cryst. 36, 944–947 (2003).

    Article  CAS  Google Scholar 

  39. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  40. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Protein Struct. Funct. Genet. 11, 281–296 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. White and K. Fütterer for help with X-ray data collection, and P. Hornbeck (Cell Signaling Technology; PhosphoSites) and F. Diella (European Molecular Biology Laboratory, Heidelberg; Phospho.ELM) for data sets of identified human phosphorylation sites. Supported by US Public Health Service (AI20963 to V.H.E. and AI33993 to D.F.H.), the Medical Research Council (B.E.W. and M.C.), the Wellcome Trust (B.E.W.), the Biotechnology and Biological Sciences Research Council (F.M.) and the Sidney Kimmel Foundation (A.L.Z.).

Author information

Author notes

  1. Fiyaz Mohammed and Mark Cobbold: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Research UK Institute for Cancer Studies, School of Cancer Sciences, University of Birmingham, Edgbaston, B15 2TT, Birmingham, UK

    Fiyaz Mohammed, Mahboob Salim & Benjamin E Willcox

  2. Carter Immunology Center and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, 22908, Virginia, USA

    Mark Cobbold, Angela L Zarling & Victor H Engelhard

  3. School of Immunity, Infection and Inflammation, University of Birmingham, Edgbaston, B15 2TT, Birmingham, UK

    Mark Cobbold

  4. Department of Chemistry, University of Virginia, Charlottesville, 22908, Virginia, USA

    Gregory A Barrett-Wilt, Jeffrey Shabanowitz & Donald F Hunt

Authors
  1. Fiyaz Mohammed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark Cobbold
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Angela L Zarling
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mahboob Salim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregory A Barrett-Wilt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeffrey Shabanowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Donald F Hunt
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Victor H Engelhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Benjamin E Willcox
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.M., M.C., A.L.Z., M.S., V.H.E. and B.E.W. did the experiments and/or analyzed the data; G.A.B.-W., J.S. and D.F.H. contributed to the mass spectrometry data and analysis; B.E.W, M.C. and V.H.E. designed the study; and B.E.W. and V.H.E. prepared the manuscript.

Corresponding author

Correspondence to Victor H Engelhard.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Tables 1–8 (PDF 5306 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mohammed, F., Cobbold, M., Zarling, A. et al. Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Nat Immunol 9, 1236–1243 (2008). https://doi.org/10.1038/ni.1660

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.1660

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing