A comprehensive mutational scan of the TAPBPR scoop loop defines a region of the sequence-activity landscape for class I MHC interactions

The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). TAPBPR features a ‘scoop’ loop that projects towards the empty MHC-I peptide binding groove and rests above the F pocket. The scoop loop is not found in the closely related homologue tapasin, and therefore may be partly responsible for the unique antigen editing properties of TAPBPR. A deep mutational scan of the TAPBPR scoop loop defines the relative effects of all single amino acid mutations on binding and peptide-mediated release of the murine H2-Dd MHC-I allomorph. Increased hydrophobic packing between the scoop loop and rim of the peptide binding groove tightens the TAPBPR-MHC-I interaction. The extracellular domains of human TAPBPR were fused to Aga2p for yeast surface display at the N-terminus, and a c-myc tag for expression detection at the C-terminus. The TAPBPR scoop loop (a.a. G27-D38) was diversified by single-site saturation mutagenesis to encode all possible 240 amino acid substitutions. The library was expressed on the yeast cell surface and sorted by fluorescence-activated cell sorting (FACS). In a first round of selection, yeast were collected that expressed TAPBPR variants that formed tight interactions to fluorescent murine H2-Dd tetramers, either in the presence or absence of two different peptides (motif and P18) that compete for MHC-I interactions and displace TAPBPR. In a second round of selection, the TAPBPR-expressing yeast that were first sorted for binding tightly to H2-Dd were now resorted for having weak interactions when competing peptides are present. The choice of two rounds of selection, the first positive and the second negative, was to mimic the biological activity of TAPBPR, which must bind empty MHC-I tightly for intracellular retention, but release the MHC-I when a high affinity peptide binds.