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

The 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 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 1/2 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 allele-specificity, 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.\n\nSignificance 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 complete view of the molecular determinants of chaperone recognition. Our data provide significant evidence that localized protein motions define the intrinsic ability of MHC-I molecules to interact with chaperones. The importance of MHC-I dynamics unifies all our findings, with broad recognition of conformationally unstable, nascent MHC-I molecules becoming restricted to a smaller set of MHC-I alleles that retain relevant dynamic motions in their folded state.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/779777v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@19da2fforg.highwire.dtl.DTLVardef@383607org.highwire.dtl.DTLVardef@63171eorg.highwire.dtl.DTLVardef@182bf14_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone\nC_LIO_LIAnalysis of s-ms timescale conformational dynamics by methyl NMR reveals allele-specific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules\nC_LIO_LIs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, \"excited state\" conformation\nC_LIO_LIRestriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane\nC_LI