Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

Abstract

Highlights Deep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone  Analysis of μs-ms timescale conformational dynamics by methyl NMR reveals allelespecific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules  μs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, "excited state" conformation  Restriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane Keywords Major Histocompatibility Complex • MHC-I • NMR spectroscopy • protein dynamics • molecular chaperone • TAPBPR • peptide editing • deep mutagenesis mapping • conformational landscape • peptide repertoire Graphical Abstract AbstractThe interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance.Here, we demonstrate that the molecular chaperone TAPBPR associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized towards one side of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α 2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α 1 /α 2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of i) chaperoning unstable MHC-I molecules at early stages of their folding process, akin to a holdase with broad allelespecificity, and ii) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, towards editing Sithe repertoire of displayed antigens. Significance StatementThe human population contains thousands of MHC-I alleles, showing a range of dependencies on molecular chaperones for loading of their peptide cargo, which are then displayed on the cell surface for T cell surveillance. Using the chaperone TAPBPR as a model, we combine deep mutagenesis with functional and biophysical data, especially solution NMR, to provide a compl...

TAPBPR Promotes Antigen Loading on MHC-I Molecules Using a Peptide Trap

“…2G ). Overall, critical sequence features of TAPBPR map to sites that scaffold the correct folded architecture of the heavy chain with β 2 m, consistent with our prior conclusions that TAPBPR chaperones nascent, improperly folded MHC-I substrates within the cell(5). Notably, the G24-R36 loop is not critical for this activity.…”

“…S4B ). No binding was observed for tetramers of a third allele, human HLA-A*01:01, consistent with TAPBPR having restricted recognition of folded pMHC-I allotypes(25). Addition of peptides increased stringency of the selections, but did not change the overall form of the mutational landscapes ( Fig.…”

“…In addition, while our solution NMR experiments provide clear evidence for a lack of a direct interaction between the TAPBPR G24-R36 loop and the MHC-I groove ( Fig. 4C ), these data were acquired on the human HLA-A*02:01 heavy chain and it remains unclear how differences in amino acid polymorphisms within the mouse H2 groove influence behavior of the TAPBPR G24-R36 loop (5, 8). While we did not find differences in the formation of empty H2-D d /TAPBPR or HLA-A*02:01/TAPBPR complexes in vitro ( Fig.…”

“…To investigate specific loop interactions with MHC-I molecules, we used solution NMR and characterized HLA-A*02:01/TAPBPR complexes prepared using either wild-type or mutant TAPBPR(5, 28). We isolated 1:1 peptide-deficient HLA-A*02:01/TAPBPR complexes where the HLA-A*02:01 heavy chain was selectively ILV proS 13 C/ 1 H methyl labeled on a 12 C/ 2 H background, whereas hβ 2 m and TAPBPR were at natural isotopic abundance.…”

“…Assembly of pMHC-I molecules occurs in the endoplasmic reticulum (ER), where the association between a peptide, the polymorphic heavy chain (human leukocyte antigen, HLA), and the invariant light chain (β 2 -microglobulin, β 2 m) is facilitated by dedicated molecular chaperones, the ER-restricted tapasin of the peptide-loading complex (PLC) and the PLC-independent TAPBPR (TAP-binding protein related) (3, 4). In addition to chaperoning MHC-I, tapasin and TAPBPR function as catalytic enhancers of peptide association and dissociation within the MHC-I groove (peptide exchange) and participate in peptide editing and quality control of assembled pMHC-I molecules (5–8). Collectively, these processes ensure the correct folding, trafficking and prolonged cell surface display of pMHC-I antigens (9).…”