Abstract
Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning), and selecting high affinity peptide cargo in the MHC-I groove (editing). While X-ray and cryoEM snapshots of MHC-I molecules prepared in complex with TAPBPR and Tapasin, respectively, have provided important insights into the empty MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 aa loop region in TAPBPR (corresponding to 11 residues in the sequence of Tapasin), which has been suggested to actively compete with incoming peptides by forming direct contacts with the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for its chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward editing of their peptide cargo are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner, as shown in MHC-I tetramer staining experiments using a TAPBPR library expressed on the surface of yeast. Detailed biophysical characterization by NMR and ITC reveal that the loop does not interact with the empty MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic “trap”. Our results suggest that, by utilizing a longer loop, TAPBPR lowers the affinity requirements for peptide selection relative to Tapasin to promote loading under conditions of reduced peptide concentration.
