A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek’s Disease

Figures

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  Fig. 1. The relative background cytokine expression in B15 and B19 SPF chickens and in different tissues of B15 and B19 at 70d-old. The relative expression levels of IFN-γ (A), IL-18 (B), and IL-10 (C) in peripheral blood were detected with dqRT-PCR method. The data came from 11 healthy B15 chickens and 6 of B19 chickens at different days old. The relative expression levels of IFN-γ (D), IL-18 (E), and IL-10 (F) in lung with respiratory tract, thymus, bursa of Fabricius, spleen and peripheral blood were detected with dqRT-PCR method. The data came from each 3 of 70 days-old B15 and B19 chickens. The experiments were independently performed twice. *, P<0.05.

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  Fig 2. Susceptibility of B15 and B19 SPF chickens to MDV Md5 and peptide preference of BF2*1501 and BF2*1901. A, The dynamic viral load of MDV Md5 in B19 and B15 haplotype chickens at 4, 7, 9, and 12 dpi. The data came from each 3 B15 and B19 chickens for each time spot. We averaged two PCR tests and subtracted the results of the mock-infected chicken samples for the viral titer calculation. B-D. Self-peptides bound to BF2*1501 and BF2*1901 as assessed by gas phase sequencing. B, Sequences of peptides bound to class I molecules isolated from red blood cells determined from peptide pools showing anchor, strong and weak signals. C, Sequences of individual peptides separated by HPLC. D, Peptide anchor residues in large letters superimposed on a wire model of class I α1 and α2 domains with those residues that are both polymorphic and potentially peptide contacts indicated as smaller letters; numbering based on HLA-A2 sequence. Single letter code for amino acids (with Φ for hydrophobic); basic residues in blue, acidic residues in red, polar residues in green, hydrophobic residues in black. Results and analysis for B15 adapted from previous study (15). E, Analysis of peptides from B19 cells…

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  Fig 3. Structural overview of BF2*1901 and the shallow and narrow peptide binding groove compared to BF2*1501. A, Superimposed overall structures of BF2*1901 complexed to MDV peptide RY8 (green), and influenza H5N1 virus M1 peptide IL9 (purple). The RMSDs of the MHC monomers were determined to be 0.533 Å for B19/RY8 versus B19/IL9. B, The alignment of α1α2 domain of B19/RY8 and B19/IL9 indicates a conformational difference in the α2 helices of the two structures. C, The peptide RY8 (green) and IL9 (purple) in the structures of BF2*1901 are presented with the 2Fo-Fc electron density maps at the 1.0 σ contour level. D, Superimposed overall structures of B19/RY8 (green) and BF2*1501 complexed to the same MDV peptide RY8 (PDB: 6LHH, blue). The RMSDs of MHC monomers were determined to be 0.645 Å for B19/RY8 versus B15/RY8. E, The electrostatic plot shows the peptide binding groove of BF2*1901 complexed to MDV peptide RY8 (green). F, The electrostatic plot shows the peptide binding groove of BF2*1501 complexed to MDV peptide RY8 (PDB: 6LHH, blue). G, The peptide RY8 in BF2*1901 is shown in green cartoon with three large B19-specific residues at the bottom of the groove (Trp95, Arg111, Tyr113)…

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  Fig 4. The different features of A and F pockets of BF2*1901 compared to BF2*1501. A, The superposing between A pockets of the two molecules M1 (green) and M2 (purple) of the asymmetrical unit of B19/RY8 structure, and also BF2*1501 complexed to MDV peptide RY8 (PDB: 6LHH, blue), flu peptide PA124 (PDB: 6IRL, yellow) and chicken calcium-binding protein peptide CBP (PDB: 6KX9, orange). The superposition clearly shows the similar conformation of P1-Arg in BF2*1901 M1 and M2, but the P1-Arg of B15/PA124 is closer to the α1 helix, while the P1-Arg of peptides RY8 and CBP are closer to the peptide itself. B, The weak hydrogen bond between P1-Arg of peptide RY8 (green) and Glu65 (green) in B19/RY8 M1. No interaction between them is observed in B19/RY8 M2 (not shown). C, The closer binding between P1-Arg in B15/PA124 (yellow) and the residues Tyr61 and Glu65 in α1 helix of BF2*1501. D, Superposition of B19/RY8 and B15/RY8 according to the α-C of α1α2 domains, clearly showing the higher position of PΩ-Tyr of peptide RY8 in B19/RY8 structure, compared to the PΩ-Tyr of peptide RY8 in B15/RY8 structure. E and F, The electrostatic plot shows the narrow and shallow F pocket of BF2*1901 (E, peptide…

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  Fig 5. The flexible but tight binding of P3 anchor of BF2*1501 compared to BF2*1901. A, The P3-Arg anchor protrudes its side chain out of the shallow and narrow D pocket of B19/RY8 groove, which is occupied by the large positively-charged residue Arg111. B, The P3-Arg anchor puts its side chain into the D pocket of B15/RY8 groove (PDB: 6LHH), which is occupied by the small residue Ser111. C, Superposition of B19/RY8 (green), B19/IL9 (purple), B15/RY8 (PDB: 6LHH, blue) and B15/PA124 (PDB: 6IRL, yellow), clearly showing two different conformations of P3 anchors of peptides presented by BF2*1501, i.e. P3-Arg in RY8 and P3-Glu in PA124. D, The smaller D pocket of B19/RY8. E, The larger D pocket of B15/RY8 and the hydrogen bond between P3-Arg and Ser111 of BF2*1501. F, The similar D pocket and P3 conformation of B19/IL9 as in B19/RY8. G, The P5-His of peptide PA124 occupies the D pocket of B15/PA124, with P3-Glu pointing out of the D pocket.

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  Fig 6. The higher α2 helix of BF2*1901/RY8 compared to BF2*1501/RY8 and BF2*1901/IL9. A, Superimposed structures of B19/RY8 (green) and B15/RY8 (PDB: 6LHH, blue), according to the Cα of α1α2 domains (residues 1-180). The different conformations of the middle portion in the α2 helices are pointed by the red arrow. B, The atomic positions of related residues of B19/RY8 (green) and B15/RY8 (blue) including the backbone atoms showed with sticks. The distance between Cα of Asp148 in the two structures was shown in red. C and D, The composite OMIT maps of α1 helices from B19/RY8 (green) and B15/RY8 (cyan). E and F, The distance measurement between the residues in the middle portion of α2 helix and the β sheets in B19/RY8 and B15/RY8. The distance between the Cα atoms of Asp148 and Thr129 in B19 (E) is longer than in B15 (F) shown in red dashed line. G and H, The two larger residues Arg111 and Tyr113 jack up the α2 helix of BF2*1901 through the interaction with Tyr149 and Trp144. The distance between the Cα atoms of Asp149 and Arg111 in B19 (G) is longer than the one between Asp149 and Ser111 in B15 (H). The hydrogen bond between Tyr149 and Arg111 is shown in black dashed line. I, Superimposed…

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  Fig 7. The weaker binding capacity of BF2*1901 compared to BF2*1501 with similar peptides. A and C, binding of peptides (A, IL9 and C, RY8) to BF2*1501 and BF2*1901 by in vitro refolding. The absorbance peak of the BF2 complex with the expected molecular mass of 45 kDa was eluted at the estimated volume of 16 mL on a Superdex 200 10/300 G L column. B and D, Thermostability of peptides (B, IL9 and D, RY8) complexed to BF2*1501 and BF2*1901 by CD spectroscopy showed by the curves generated from the raw data. The TmS of different complexes are indicated by the temperature when 50% of the protein unfolded at the black dashed line. The experiments were independently performed twice.