Figure 4. Residues K22-D35 are essential for soluble TAPBPR to bind peptide-loaded MHC I.

(a and c) Histograms of soluble TAPBPR loop variant binding to HeLaM-HLA-ABC^KO cells expressing HLA-A*68:02 incubated with 100 nM TAPBPR at (a) 37°C or (c) 26°C for 30 min. TAPBPR^TN5, a TAPBPR variant which cannot bind to MHC I, is included as a negative control. (b) TAPBPR pull-downs on IFNγ-treated HeLaM-TAPBPR^KO cells incubated with soluble TAPBPR loop mutants reveal all variants are capable of binding to MHC I, but do not bind to UGT1. TAPBPR^TN5 is included as a non-MHC binding control. Data is representative of three independent experiments. (d) Bar graph comparing soluble TAPBPR variant binding to HeLaM-HLA-ABC^KO+A*68:02 cells at 37°C with 26°C from three independent experiments. Error bars represent -/+SD. (e) Histograms show typical fluorescent peptide binding to IFNγ induced HeLaM cells treated -/+100 nM soluble TAPBPR variants for 15 min at 26°C, followed by incubation with 10 nM ETVSK*QSNV for 15 min at 26°C. (f) Bar graph compares ETVSK*QSNV peptide binding to HeLaM cells treated -/+ soluble TAPBPR variants at 37°C with 26°C from three independent experiments. Error bars represent -/+SD. n/s = not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, using unpaired two-tailed t-tests.

Figure 4—figure supplement 1. Pre-incubation with high affinity peptide inhibits TAPBPR^Øloop binding to HLA-A*68:02 molecules at 26°C.

HeLaM-HLA-ABC^KO cells reconstituted with HLA-A*68:02 were incubated at 26°C for 12 hr, then subsequently treated -/+1 µM ETVSK*QSNV peptide for 30 min at 26°C, followed by washing twice in PBS to remove unbound peptide. Cells were then incubated cells with -/+100 nM TAPBPR^WT or TAPBPR^Øloop at 26°C for 15 min, washed in PBS, then cooled to 4°C for detection of bound TAPBPR using the mAb PeTe4. (a) Histograms show the fluorescent peptide binding to the cells incubated at 26°C used to test soluble TAPBPR^WT and TAPBPR^Øloop binding. (b) Histograms show the typical TAPBPR^WT and TAPBPR^Øloop binding to cells incubated at 26°C with (red line) and without (black line) pre-incubation with the HLA-A*68:02-binding peptide. (c) Bar chart show the MFI of TAPBPR binding from three independent experiments. Error bars represent -/+SD. The data show that pre-incubation of cells with high-affinity peptide significantly reduces the ability of soluble TAPBPR^Øloop to bind to HLA-A*68:02 (***p≤0.001) but does not significantly inhibit the ability of TAPBPR^WT to bind to HLA-A*68:02.