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

Abstract

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 TAP-binding protein related (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 toward the α2domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2domain. 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 α1/α2helices 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 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) 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, toward editing the repertoire of displayed antigens.

Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology

“…In our own prior studies, deep mutagenesis of human membrane proteins was used to understand how sequence relates to folding based on surface localization, as misfolded sequences were retained intracellularly due to quality control machinery [50,51,79]. However, PDGFRα is an exception, and nearly every missense mutation still reaches the plasma membrane, including those that are incompatible with the native folded structure (for example, mutations that disrupt disulfides, create cavities, impose steric clashes, or introduce buried charges).…”

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

“…S10A-H), demonstrating that the TAPBPR G24-R36 loop employs a similar mechanism to promote peptide capture irrespective of peptide length. DISCUSSION Recent structural studies have revealed that chaperone-mediated peptide exchange/editing is achieved through (i) stabilization of the peptide-deficient MHC-I in a peptide-receptive conformation, (ii) ejection of suboptimal peptides by inducing an ~3 Å widening of the MHC-I groove, and (iii) regulation of a dynamic switch located in the MHC-I groove (Jiang et al, 2017;McShan et al, 2018McShan et al, , 2019Thomas and Tampé, 2017). Despite these important findings, the mechanistic details of how chaperones influence the repertoire of MHC-I presented antigens remains incompletely characterized, while a significant difference between published X-ray structures challenge the proposed role of the TAPBPR G24-R36 loop as a direct peptide competitor.…”

“…Solution NMR experiments provide clear evidence for a lack of a direct interaction between the TAPBPR G24-R36 loop and the HLA-A*02:01 groove (Fig. 4C), however in vitro binding data were acquired using the human HLA-A*02:01 heavy chain and it remains unclear how differences in amino acid polymorphisms within other MHC-I, such as the mouse H2 groove, influence behavior of the TAPBPR G24-R36 loop (McShan et al, 2019;Morozov et al, 2016). While we did not find differences in the formation of empty H2-D d /TAPBPR or HLA-A*02:01/TAPBPR complexes in vitro by SEC ( Fig.…”

“…Bound peptide, hβ2m or TAPBPR were fully protonated. NMR methyl resonance assignments of free and TAPBPR bound states of HLA-A*02:01 were reported previously by our group (McShan et al, 2019). Peptide-deficient HLA-A*02:01/hβ2m/TAPBPR complexes were prepared as described above in the section "Preparation of empty MHC-I/TAPBPR complexes" where HLA-A*02:01 was ILV proS or AILV methyl labeled.…”

“…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 (β2-microglobulin, β2m) 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) (van Hateren et al, 2017;Hermann et al, 2015a). 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 (Fleischmann et al, 2015;Hermann et al, 2015b;McShan et al, 2019;Morozov et al, 2016). Additional quality control is achieved by the association of TAPBPR with UDPglucose:glycoprotein glucosyltransferase (UGGT1), which promotes reglycosylation of empty or suboptimally loaded MHC-I towards their recycling to the PLC Zhang et al, 2011).…”