A deep mutational scan of the MHC class I-specific chaperone TAPBPR for sites important for peptide editing

TAP-binding protein-related (TAPBPR) is an endoplasmic reticulum-resident chaperone that facilitates class I MHC (MHC-I) processing and peptide loading. TAPBPR has (1) chaperone function to stabilize misfolded or partially folded nascent MHC-I substrates, and (2) editing function, in which it catalyzes the exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. TAPBPR-TM is a chimera of the TAPBPR ectodomain and a canonical TM domain, which escapes the endoplasmic reticulum to reach the cell surface. We reasoned that at the cell surface, TAPBPR-TM is more likely to interact with folded MHC-I and thus surface interactions will be more representative of editing function. When tapasin, a homolog of TAPBPR and the main MHC-I-specific chaperone, is knocked out, surface trafficking of HLA-A2 (a human MHC-I allele) is severely diminished, but surface HLA-A2 levels are rescued by over-expression of TAPBPR-TM. Using this assay as the foundation for a fluorescence-based selection, we deep mutationally scanned 104 positions on TAPBPR-TM to identify sites critical for engaging folded MHC-I. A chimeric fusion was constructed between the extracellular domains of human TAPBPR and the transmembrane helix of HLA-G. The chimera, called TAPBPR-TM, has higher activity for functional replacement of tapasin than wild type TAPBPR. A library was constructed in TAPBPR-TM in which 104 positions where diversified with degenerate NNK codons. The library was transfected in to tapasin-knock out Expi293F cells after 1500-fold dilution with a carrier plasmid; this ensures most cells express no more than a single sequence variant and there is a tight connection between genotype and phenotype. Cells expressing the TAPBPR-TM library were stained for surface HLA-A2 with R-phycoerythrin-conjugated anti-HLA-A2 clone BB7.2, and the 0.5% of cells with the highest fluorescence were collected by FACS. Following Illumina sequencing, the frequencies of mutations from transcripts in the sorted cells were compared to their respective frequencies in the naive plasmid library. The calculated enrichment ratios are a proxy for relative activity of the TAPBPR-TM sequence variants.