A deep mutational scan of the MHC class I-specific chaperone tapasin
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PROVIDER: PRJNA668133 | ENA |
REPOSITORIES: ENA
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Project description:Tapasin acts as the principal MHC-I-specific chaperone for facilitating folding and antigenic peptide loading of nascent MHC class I substrates in the cell. In cells where tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of tapasin rescues HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, tapasin was deep mutationally scanned at 108 positions in the core, at the interface with MHC-I, and on the ‘backside’ distal from where MHC-I binds. Critical residues of tapasin for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain, to the surface contacting the MHC-1 beta2m and alpha-3 domains, and to the base of a protruding loop that rests above the peptide-binding groove (but not to the tip of the loop itself).
2020-10-09 | GSE159247 | GEO
Project description:The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). TAPBPR features a ‘scoop’ loop that projects towards the empty MHC-I peptide binding groove and rests above the F pocket. The scoop loop is not found in the closely related homologue tapasin, and therefore may be partly responsible for the unique antigen editing properties of TAPBPR. A deep mutational scan of the TAPBPR scoop loop defines the relative effects of all single amino acid mutations on binding and peptide-mediated release of the murine H2-Dd MHC-I allomorph. Increased hydrophobic packing between the scoop loop and rim of the peptide binding groove tightens the TAPBPR-MHC-I interaction.
2019-10-31 | GSE118568 | GEO
Project description:TAP-binding protein-related (TAPBPR) is an endoplasmic reticulum-resident chaperone that facilitates class I MHC (MHC-I) processing and peptide loading. TAPBPR has (1) chaperone function to stabilize misfolded or partially folded nascent MHC-I substrates, and (2) editing function, in which it catalyzes the exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. TAPBPR-TM is a chimera of the TAPBPR ectodomain and a canonical TM domain, which escapes the endoplasmic reticulum to reach the cell surface. We reasoned that at the cell surface, TAPBPR-TM is more likely to interact with folded MHC-I and thus surface interactions will be more representative of editing function. When tapasin, a homolog of TAPBPR and the main MHC-I-specific chaperone, is knocked out, surface trafficking of HLA-A2 (a human MHC-I allele) is severely diminished, but surface HLA-A2 levels are rescued by over-expression of TAPBPR-TM. Using this assay as the foundation for a fluorescence-based selection, we deep mutationally scanned 104 positions on TAPBPR-TM to identify sites critical for engaging folded MHC-I.
2023-03-09 | GSE218828 | GEO
Project description:A comprehensive mutational scan of the TAPBPR scoop loop defines a region of the sequence-activity landscape for class I MHC interactions
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Project description:The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). TAPBPR features a loop (amino acids 24-35) that projects towards the empty MHC-I peptide binding groove and rests above the F pocket. The 24-35 loop is much shorter in the closely related homologue tapasin, and therefore may be partly responsible for the unique antigen editing properties of TAPBPR. Previously we reported a deep mutational scan of human TAPBPR focused on the 24-35 loop, and determined the relative effects of single amino acid mutations on binding and peptide-mediated release of the murine H2-Dd MHC-I allomorph. Here, we extend our studies to determine the mutational landscape of the 24-35 loop when TAPBPR binds a human MHC-I allomorph, HLA-A*02:01. The data highlights how TAPBPR affinity can be increased or decreased for different MHC-I allomorphs by tuning the electrostatic complementarity of the 24-35 loop for surfaces on the rim of the peptide-binding groove. By changing the selection pressure from HLA-A2 binding to HLA-A2 loading and processing, we find that TAPBPR is reasonably tolerant of mutations in the 24-35 loop for efficient peptide-MHC-I processing and surface trafficking.
2019-09-01 | GSE126206 | GEO
Project description:TAP-binding protein-related (TAPBPR) facilitates the processing of nascent class I MHC (MHC-I) by (1, chaperone function) acting as a chaperone for misfolded or partially folded MHC-I substrates in the cell and (2, editing function) by catalyzing exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. In cells in which the principal MHC-I-specific chaperone and TAPBPR homologue tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of TAPBPR can partially replace tapasin and rescue HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, TAPBPR was deep mutationally scanned at 92 positions in the core, at the interface with MHC-I, and on the ‘backside’ distal from where MHC-I binds. Critical regions of TAPBPR for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain and residues that contact the junction between beta-2 microglobulin and alpha-3 domains. Key sites on TAPBPR for chaperone activity therefore scaffold assembly of the MHC-I H chain with beta-2 microglobulin.
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