Figure 1.
pBL50 values obtained with N*01301 using an RMA-S stabilization assay, (see Materials and Methods for explanation), (A) for the reference peptide (TIMPKDIQL) and Ala-substituted peptides and (B) forTp1214–224 (VGYPKVKEEML) and Ala- substituted peptides.
Figure 2.
Overview of the structure of N*01301.
(A) The heavy chain is shown in cyan, β2m in purple, and the peptide in yellow (B) Top view of the binding platform showing the bound peptide Tp1214–224 in stick format with residues labeled.
Figure 3.
Conformation of the bound peptide.
Conformation of the bound peptide Tp1214–224 shown from the α2 helix side of the binding groove, unless stated otherwise. (A) The corresponding 2Fo-Fc electron density is shown in mesh format. The peptide is in stick format with each amino acid labeled (B) View onto the binding groove (as in Fig. 2B) additionally showing Trp3 and Glu9 of Tp1214–224, and Arg155 of N*01301. Hydrogen bonds are denoted by dotted grey lines (C) The peptide backbone is shown as a cartoon ribbon in gold. The Lys at position 5 of Tp1214–224 and the Asn, Glu and Trp at positions 70, 97 and 73 respectively of N*01301 are shown in stick format.
Figure 4.
Peptide Tp1214–224 bound to N*01301 (cyan), compared to peptides in other mammalian pMHC structures. View shows the Cα-traces of the bound peptide from the α2 helix side of the binding groove, unless stated otherwise. (A) Long peptide comparison: 11mer peptide (green, PDB accession code 3BEV) bound to chicken B21; 11mer (gold, PBD code 2FZ3), 12mer (red, PBD code 3BW9) and 13mer (orange, PBD code 1ZHL) peptides bound to HLA-B*3508; 11mer peptides (purple, PBD code 2BVQ; grey, PBD code 2BVO) bound to HLA-B*5703. (B) Long peptide comparison: view onto the binding groove of Fig. 4A. (C) Long peptide comparison: 11mer peptide bound to HLA-B*3508 (blue, PDB code 1ZSD), and its conformation change upon TCR binding (pink, PDB code 2NX5). Peptide Cα backbone positions are denoted by P1-PΩ for Tp1214–224. (D) Mouse comparison: 9mer peptide (red, PBD code 1CE6) and 11mer peptide (blue, PBD code 1JPF) bound to H-2Db; 10mer peptide (brown, PBD code 1BII) bound to H-2Dd; 8mer peptide (green, PBD code 1FO0) bound to H-2Kb; 9mer peptide (pink, PBD code 1VGK) bound to H-2Kd; 9mer peptide (yellow, PBD code 1ZT1) bound to H-2Kk. (E) Mouse comparison: view onto the binding groove of Fig. 4D.
Table 1.
Amino acid residues at positions 73 and 147 in 7 cattle MHC class I alleles.
Figure 5.
Effects of single alanine substitutions in the Tp1214–224 epitope on recognition by specific CD8+ T cell clones.
T cell recognition was examined using a 4-hour 111Indium release cytotoxicity assay employing target cells incubated with 1 ug/ml of peptide. The maximum level of cytotoxicity obtained with the native Tp1214–224 peptide is shown for each clone. Killing of unpulsed target cells was <2% for all clones. The degree of reduction in killing obtained with each of the single Ala substituted peptides (A1–A11) compared to that obtained with the native Tp1214–224 peptide is represented as follows: 0–20% reduction (white); 21–40% reduction (pale grey); 41–60% reduction (medium grey); 61–80% reduction (dark grey); 81–100% reduction (black). The expressed TCR Vβ genes were identified using Vβ subfamily-specific PCR assays. NT = not tested.