Sites on TAPBPR that scaffold assembly of a MHC-I H chain with beta2m are important for TAPBPR chaperone activity

TAP-binding protein-related (TAPBPR) facilitates the processing of nascent class I MHC (MHC-I) by (1, chaperone function) acting as a chaperone for misfolded or partially folded MHC-I substrates in the cell and (2, editing function) by catalyzing exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. In cells in which the principal MHC-I-specific chaperone and TAPBPR homologue tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of TAPBPR can partially replace tapasin and rescue HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, TAPBPR was deep mutationally scanned at 92 positions in the core, at the interface with MHC-I, and on the 'backside' distal from where MHC-I binds. Critical regions of TAPBPR for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain and residues that contact the junction between beta-2 microglobulin and alpha-3 domains. Key sites on TAPBPR for chaperone activity therefore scaffold assembly of the MHC-I H chain with beta-2 microglobulin. Overall design: A chimeric fusion was constructed between the extracellular domains of human TAPBPR and the transmembrane and cytosolic regions of tapasin. The chimera, called TAPBPR-CT, has higher activity for functional replacement of tapasin than wild type TAPBPR, possibly because of recruitment to the TAP1/2 transporter mediated by the tapasin C-terminal tail. A library was constructed in TAPBPR-CT in which 92 positions were 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. Illumina sequencing data to validate the introduction of indel mutations in exon 2 of the tapasin gene is in GEO Series Acc. No. GSE126206. Cells expressing the TAPBPR-CT 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 sequence variants.