Fig. 6. NMR characterization of chaperone-mediated peptide exchange on MHC-I using selective methyl ¹³C-labeled peptides.

a Rosetta model of the TAX9/HLA-A*02:01/TAPBPR complex using template PDB ID 5OPI. The Leu/Val methyl groups of TAX9 are shown as spheres. HLA-A*02:01 is green, TAX9 is magenta, TAPBPR is gray, and the TAPBPR “scoop loop” is colored salmon. b 2D ¹H-¹³C methyl HMQC spectra of 100 μM ¹⁵N/¹³C LV-labeled TAX9 peptide in the free state (left, green), in complex with HLA-A*02:01/hβ₂m (middle, blue), and in complex with HLA-A*02:01/hβ₂m in the presence of saturating concentration (eightfold molar excess) TAPBPR (right, purple). All spectra were recorded at 25 °C at a ¹H field of 800 MHz. Assignments of methyl resonances corresponding to the free and MHC-bound peptide states are denoted with ‘f’ and ‘b’, respectively c Rosetta model of the TAX9/HLA-A*02:01/TAPBPR complex using template PDB ID 5WER is shown highlighting that TAPBPR promotes the release of TAX9 peptide. The open, intermediate, and closed conformations of the TAPBPR G24-R36 loop obtained from MD simulations are shown in gray, salmon, and yellow. d Overlay of 2D ¹H-¹³C methyl HMQC spectra from ‘A’ of: ¹³C-LV-labeled TAX9 in the free state (green), and in complex with HLA-A*02:01/hβ₂m in the presence of saturating concentration of TAPBPR (red). e Comparison of signal intensities for methyl resonances of TAX9 when released from the MHC-I/TAPBPR complex formed with and without the TAPBPR G24-R36 loop. f Rosetta model of disulfide-linked CysTAX9/HLA-A*02:01 in complex with TAPBPR designed using Disulfide by Design v2. The disulfide is formed between C1 of CysTAX9 (CLFGYPVYV) and W167C of HLA-A*02:01. The open and closed conformations of the TAPBPR G24-R36 loop obtained from MD simulations are shown in gray, salmon and yellow. g 2D ¹H-¹³C methyl HMQC spectra of ¹³C-LV-labeled CysTAX9 in complex with HLA-A*02:01/hβ2m (red) and HLA-A*02:01/ hβ2m in the presence of 8-fold molar excess TAPBPR (blue) recorded at 25 °C at a ¹H field of 600 MHz. Methyl resonances of the pMHC-I bound state are denoted “b” for bound. Methyl resonances of the pMHC-I/TAPBPR state are denoted “i” for intermediate, corresponding to a free peptide conformation that is still tethered to the MHC groove. h Comparison of NMR signal intensities for each methyl resonance of CysTAX9 in the pMHC-I/TAPBPR intermediate state, formed with and without the TAPBPR G24-R36 loop. The intermediate states for the Valines were not assigned. i NMR-based model of how the TAPBPR G24-R36 loop influences peptide loading on pMHC-I. j Two putative mechanisms for how the TAPBPR G24-R36 loop promotes the early capture of the peptide within the MHC-I groove with intermediate annealing from the N-terminus (left) or C-terminus (right). Data presented in panels (e) and (h) are mean ± SD for n = 3 independent experimental replicates.