Fig. 2: Important chaperone features for functional replacement of tapasin localize to scaffolding sites for MHC-I.

a TAPBPR and variants of tapasin were transfected in tapasin-KO Expi293F cells, and relative surface HLA-A*02:01 expression as measured by flow cytometry is plotted. Data are mean ± SD, n = 4 independent experiments. Proteins were FLAG-tagged at their luminal N-termini and immunoblots of the whole lysate are shown, with cyclophilin B (CycB) used as a loading control. b Tapasin was deep mutationally scanned based on selection of tapasin-KO cells with rescued surface HLA-A*02:01. Log₂ enrichment ratios for mutations across residues 10–20 are plotted from ≤−3 (depleted/deleterious, orange) to ≥+3 (enriched, dark blue). Residue position is on the horizontal axis, and amino acid substitutions are on the vertical axis (*, stop codon). c Conservation scores from the entire deep mutational scan of the tapasin/MHC-I interface are mapped to a homology model of HLA-A*02:01-bound tapasin. Highly conserved residues for mediating the folding and surface trafficking of HLA-A*02:01 are colored orange, while neutral regions are pale white/blue. Residues excluded from the library and analysis are gray. MHC-I H chain and hβ₂m are pale and dark green ribbons, respectively. d A chimera of the TAPBPR luminal domain with the tapasin TM and cytosolic domains, called TAPBPR-CT, has increased activity for chaperoning endogenous HLA-A*02:01, despite reduced protein expression by immunoblot (upper inset). Variants of TAPBPR were tested for rescue of surface HLA-A*02:01 in the TAPBPR-CT background. Data are mean ± SD, n = 4 independent experiments. e As in (b), now plotting log₂ enrichment ratios for mutations in TAPBPR residues 24–35 from a deep mutational scan for rescue of surface HLA-A*02:01 in TAPBPR library-transfected tapasin-KO cells. f As in (c), now showing sequence conservation from a deep mutational scan of TAPBPR mapped to a model of TAPBPR/HLA-A*02:01. g Close up views of two structural regions, colored as in (f). Accompanying heatmaps plot log₂ enrichment ratios for each mutation from depleted/deleterious (orange) to neutral (white and pale colors) to enriched (dark blue). h Individual mutations of TAPBPR-CT were validated by targeted mutagenesis in the tapasin surrogate assay. Data are mean ± SD, n = 4 independent experiments.