Fig. 1. Divergent TAPBPR sequences show differences in stability and recognition of HLA allotypes.

(A) Phylogenetic analysis of TAPBPR ectodomain sequences from selected species. Evolutionary analysis was conducted in MEGA7. The bootstrap values (500 bootstrap trials), depicted as spheres, indicate the percentage of trees in which the associated taxa clustered together. (B) Surface representation of hTAPBPR in contact with mouse H2-D^d/hβ₂m complex (PDB ID: 5WER) (left) (24). The predicted contact residues of hTAPBPR are highlighted in purple or red for the conserved or polymorphic residues between human and chicken orthologs, respectively (right). For the polymorphic sites, the respective chicken residues are shown in red. (C) Comparison of the electrostatic potential surfaces for hTAPBPR and chTAPBPR in the indicated ranges (down to −70 k_BT/e in red and up to +70 k_BT/e in blue, where k_B is the Boltzmann constant, T is the temperature, and e is the unit charge) as calculated using the Adaptive Poisson-Boltzmann Solver (APBS) solver in PyMOL (74). The structural model of chTAPBPR was generated using the BAKER-ROBETTA server (72). (D) T_m (in degrees Celsius) obtained from DSF for recombinant hTAPBPR, chTAPBPR, and moTAPBPR. The data shown represent replicate assays (n = 3). (E) Native gel shift analysis of TAPBPR orthologs and MHC-I complex formation. Each well is loaded with pMHC-I (A02, HLA-A*02:01 refolded with KILGFVFJV; A24, HLA-A*24:02 refolded with VYGJVRACL; and A01, HLA-A*01:01 refolded with STAPGJLEY), where J = 3-amino-3-(2-nitrophenyl)-propionic acid, TAPBPR, or 1:1 molar ratio mixture. Samples were UV-irradiated for 1 hour at 365 nm, when indicated. The formed MHC-I/TAPBPR complexes are indicated with arrows.