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We and others have demonstrated that ERAP1 processes peptides bound to MHC I, but the underlying mechanism is unknown. To this end, we utilized single-chain trimers (SCT) of the ovalbumin-derived epitope SIINFEKL (SL8) tethered to the H2-K<sup>b</sup> MHC I determinant from mouse and introduced three substitutions, E63A, K66A, and W167A, at the A-pocket of the peptide-binding groove in the MHC I heavy chain, which interact with the N termini of peptides. These variants significantly decreased SL8-presenting SCT at the cell surface in the presence of ERAP1, but did not affect overall SCT expression, indicating that ERAP1 trims the SL8 N terminus. Comparison of the X-ray crystal structures of WT and three variant SCTs revealed only minor perturbations of the peptide-binding domain in the variants. However, molecular dynamics simulations suggested that SL8 can dissociate partially within a sub-microsecond timescale, exposing its N terminus to the solvent. We also found that the C terminus of MHC I-bound SL8 remains deeply buried in the F-pocket of MHC I. Furthermore, free-energy calculations revealed that the three SCT variants exhibit lower free-energy barriers of N terminus dissociation than the WT K<sup>b</sup> Taken together, our results are consistent with a previously observed model in which the partial dissociation of bound peptides from MHC I exposes their N terminus to trimming by ERAP1, whereas their C terminus is anchored at the F-pocket.","bibjson":{"author":[{"initials":"A","lastname":"Papakyriakou","name":"Papakyriakou A"},{"initials":"E","lastname":"Reeves","name":"Reeves E"},{"initials":"M","lastname":"Beton","name":"Beton M"},{"initials":"H","lastname":"Mikolajek","name":"Mikolajek H"},{"initials":"L","lastname":"Douglas","name":"Douglas L"},{"initials":"G","lastname":"Cooper","name":"Cooper G"},{"initials":"T","lastname":"Elliott","name":"Elliott T"},{"initials":"JM","lastname":"Werner","name":"Werner JM"},{"initials":"E","lastname":"James","name":"James E"}],"identifier":[{"id":"10.1074/jbc.RA117.000313","type":"doi"},{"id":"29599287","type":"pubmed"}],"issue":[null],"journal":{"iso_abbreviation":"J. Biol. Chem.","name":""},"pages":[null],"title":"The partial dissociation of MHC class I bound peptides exposes their N terminus to trimming by endoplasmic reticulum aminopeptidase 1.","type":"article","url":"https://www.sciencedirect.com/science/article/abs/pii/S0021925820369556","volume":[null],"year":[2018]},"in_pmc":"N","in_pmce":"Y","open_access":"Y"},"resolution":"2.40","same_as":{"pdbe":{"url":"https://www.ebi.ac.uk/pdbe/entry/pdb/5oqi"},"rcsb":{"url":"https://www.rcsb.org/structure/5oqi"}},"species":{"common_name":"Mouse","match_type":"histo:assign_species","scientific_name":"Mus musculus","slug":"mus_musculus"},"tcr":null,"title":"Single chain construct of H2-Kb binding \"None\" at 2.40&#8491; resolution","unique_chain_count":1}}