Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes

Abstract

Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

“…We further hypothesized that the interchain disulfide engineering could be applied to different HLA allotypes resulting in a universal, open MHC-I platform for antigen screening experiments. To quantitatively compare peptide exchange rates across different alleles, we then performed a series of FP experiments using optimized placeholder peptides, pHLA concentrations, and the protocol(26), where the binding of high-affinity fluorophore-labeled peptides was monitored through an increase in polarization ( Fig. S6 ).…”

“…While tapasin has shown preferential binding to HLA-B alleles, TAPBPR preferably interacts with HLA-A alleles but mainly covers the A02 and A24 supertypes (46,63). More recent work has expanded the TAPBPR-mediated peptide exchange on a broad repertoire of allotypes using TAPBPR orthologs and engineered variants (26). However, compared to the open MHC-I platform, the approach requires optimized placeholder peptides and recombinant chaperone proteins to stabilize empty, receptive molecules.…”

“…We also found low deuterium exchange on regions corresponding to the α1 helix and β-sheet floor of the peptide binding groove for the loaded molecules. Previous studies for WT HLA-A*02:01 have established that the α2-1 helix shows high deuterium uptake in the empty state (26,54), likely due to an approximate 3 Å widening of the groove seen in crystal structures (13,45,55). In contrast, our HDX data recorded for open HLA-A*02:01 showed that residues 140-159 on the α2-1 helix have a similar level of deuterium uptake between the peptide-loaded and empty molecules (Fig.…”

“…The copyright holder for this preprint this version posted March 18, 2023. ; https://doi.org/10.1101/2023.03.18.533266 doi: bioRxiv preprint of FP experiments using optimized placeholder peptides, pHLA concentrations, and the protocol (26), where the binding of high-affinity fluorophore-labeled peptides was monitored through an increase in polarization (Fig. S6).…”

“…Conditional ligands, bound to the MHC-I, can be cleaved by UV exposure or released by increasing the temperature to generate empty molecules, which can be loaded with a rescuing peptide (19)(20)(21). Dedicated MHC-I chaperones, including TAPBPR and its orthologs, have also been used to stabilize an array of different MHC-I allotypes in a peptide-receptive conformation with a preferred binding to HLA-A over HLA-B and HLA-C alleles, promoting the exchange of low-to intermediate-affinity peptides for high-affinity peptides in a process known as "peptide-editing" (22)(23)(24)(25)(26). In addition, molecular dynamics (MD) simulations combined with a tryptophan fluorescence assay have shown that empty MHC-I molecules are not molten globules like previously reported (27), but have varying degrees of structure in the α1 and α2 helices (28,29).…”