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.

Monoclonal antibodies targeting the SARS‐CoV‐2 spike (S) neutralize infection and are efficacious for the treatment of COVID‐19. However, SARS‐CoV‐2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS‐CoV‐2 variants and related betacoronaviruses. The high‐affinity and catalytically active decoy sACE2 2 .v2.4‐IgG1 was previously shown to be effective against SARS‐CoV‐2 variants when administered intravenously. Here, inhalation of aerosolized sACE2 2 .v2.4‐IgG1 increased survival and ameliorated lung injury in K18‐hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE2 2 .v2.4‐IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18‐hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE2 2 .v2.4‐IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS‐CoV‐2 variants.

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

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...

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22.v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE22.v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE22.v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE22.v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.