Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

ACCEPTED MANUSCRIPT Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove Curtis McMurtrey, Thomas Trolle, Tiffany Sansom, Soumya G Remesh, Thomas Kaever, Wilfried Bardet, Kenneth Jackson, Morten Nielsen, Rima McLeod, Dirk M Zajonc, Ira J Blader, Bjoern Peters, Alessandro Sette, William Hildebrand DOI: http://dx.doi.org/10.7554/eLife.12556 Cite as: eLife 2016;10.7554/eLife.12556 Received: 26 October 2015 Accepted: 28 January 2016 Published: 29 January 2016 This PDF is the version of the article that was accepted for publication after peer review. Fully formatted HTML, PDF, and XML versions will be made available after technical processing, editing, and proofing. Stay current on the latest in life science and biomedical research from eLife. Sign up for alerts at elife.elifesciences.org 1 2 Toxoplasma gondii Peptide Ligands Open the Gate of the HLA Class I Binding Groove 3 4 5 6 Curtis McMurtrey1,2, Thomas Trolle3,4 , Tiffany Sansom5, Soumya G. Remesh4, Thomas Kaever4, Wilfred Bardet1, Kenneth Jackson1, Morten Nielsen3,6, Rima McLeod7, Dirk M. Zajonc4, Ira J. Blader5, Bjoern Peters4, Alessandro Sette4 William Hildebrand1,2. 8 1. University of Oklahoma Health Science Center, Department of Microbiology and Immunology, Oklahoma City, OK, USA 9 2. Pure MHC LLC, Austin, TX, USA 10 11 3. Center for Biological Sequence Analysis, Technical University of Denmark, Kgs. Lyngby, Denmark 12 4. La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA 13 5. University at Buffalo School of Medicine, Department of Microbiology and Immunology, Buffalo, NY, USA 7 14 16 6. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina 17 7. University of Chicago, Chicago, IL, USA 15 18 19 Corresponding Author: 20 21 22 23 24 25 William Hildebrand University of Oklahoma Health Science Center Biomedical Research Center, Room 317 975 NE 10th st. Oklahoma City, Oklahoma, 73104 26 27 28 1 29 Abstract 30 HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell 31 recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to 32 peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes 33 from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 34 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. 35 Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these 36 longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C- 37 terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of 38 extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to 39 protrude through one end of the binding groove. In summary, we demonstrate that unrealized 40 structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique 41 C-terminal peptide extensions. 42 43 44 Impact Statement 45 T. gondii infection alters the presentation of peptide ligands to the immune system by inducing 46 a previously unreported structural change in the HLA class I binding groove. 47 2 48 Introduction 49 CD8 T-cells mediate immunity to Toxoplasma gondii infection (1, 2) through recognition of 50 peptide antigens presented by the MHC class I (MHC I) molecules of infected cells (3, 4). The 51 majority of peptide ligands identified to date are derived from parasite surface proteins, 52 proteins localized to dense granules, or the rhoptry proteins which are specialized secretory 53 granules whose contents are released either into the host cell cytoplasm or the 54 parasitophorous vacuole (5-7). These secreted proteins are thought to be optimal candidates 55 for MHC I presentation because they have the best access to conventional antigen processing 56 and presentation machinery in the host cell. However, this is a large pathogen, and the full 57 array of parasite proteins that might be sampled and presented remains unknown. 58 Recent advances in immunology and proteomics highlight that non-canonical ligands are 59 presented to T cells by MHC I molecules. While a majority of peptides are 8-11 amino acids in 60 length, MHC I molecules present a considerable number of peptides >11 amino acids (8, 9) that 61 elicit T-cell responses (8, 10). Structural characterizations suggest that these long ligands 62 interact with the MHC I molecule much like canonical peptides: The MHC I alpha chain forms a 63 10 x 25 angstrom groove in which peptide ligands are anchored by their second (P2) and C- 64 terminal (PΩ) residues. In this mode of binding, the middle portion of any oversized peptides 65 can bulge out of the MHC I groove and interact with the receptors of T lymphocytes (11). 66 Crystallographic studies have confirmed this bulging model, although there exists a structural 67 example of a 10mer interacting with MHC I molecule HLA-A2 via P2 and P9 with an amino acid 68 extension at P10 (12). Thus, both peptide extension and peptide bulging have been observed 3 69 for MHC I ligands, and, as longer ligands become increasingly evident, the interaction of these 70 ligands with MHC I will need to be clarified. 71 The goal of this study was to have the MHC I of infected cells inform the number, breadth, and 72 nature of T. gondii peptide ligands. HLA-A*02:01 was purified from cells infected with T. gondii 73 and peptide ligands eluted from the HLA class I (human MHC I) complex were analyzed by two- 74 dimensional LCMS. The resulting data demonstrate that nearly 200 ligands originating from 75 close to 100 different T. gondii proteins are sampled for MHC I presentation. As envisioned, a 76 number of ligands originating from dense granule proteins was observed (5, 7), yet MHC I 77 ligands were also derived from a large number parasite cytoplasmic proteins. Surprisingly, T. 78 gondii ligands were significantly longer than existing structural models can accommodate, and a 79 series of peptide analogs demonstrated that these longer peptides are not anchored to MHC I 80 via their C-termini. Crystallographic studies reveal an unreported structural re-arrangement of 81 residues in the MHC I binding groove that accommodate C-terminal peptide extensions, and 82 this structural flexibility is discussed in the context of infection by intracellular pathogens. 83 Results 84 Identification of Toxoplasma gondii HLA-A*02:01 Ligands 85 The first objective of this study was to identify pathogen-encoded ligands made available by 86 MHC I. To accomplish this objective, HLA-A*02:01 was purified from T. gondii infected THP-1 87 monocytes as described. (13, 14). To ensure THP-1 cells were infected, the number of infected 88 cells and (...truncated)