The partial dissociation of MHC class I–bound peptides exposes their N terminus to trimming by endoplasmic reticulum aminopeptidase 1

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 process N-terminally extended antigenic precursors for optimal loading onto major histocompatibility complex class I (MHC I) molecules. 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-Kb 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 Kb. 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.

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

“…A recent study demonstrated ERAP1mediated peptide trimming of a 16mer peptide that was anchored onto the MHCI via a disulfide bond near its C-terminus (23). In this case, ERAP1 trimming cannot possibly follow peptide full dissociation since the peptide is covalently bound onto the MHCI, but the peptide Nterminus could transiently detach from the Apocket and extend away from the MHCI as previously modelled (31). A long overhang could be structurally consistent with ERAP1 trimming, but steric hindrance and lack of activation by the regulatory site should make trimming less efficient.…”

“…This was recently demonstrated with a series of extended peptides bound onto HLA-B08 (37). In contrast, formation of a transient ERAP1/MHCI trimming complex would necessitate conformations not before observed experimentally, but simulated computationally (31,51). To date, no direct ERAP1/MHCI protein-protein interactions have been demonstrated (26).…”

“…In addition, a specific interaction between the C terminus of the peptide and a regulatory site of ERAP1 was found to be important for longer peptides (49). In contrast, on-MHCI trimming necessitates that at least some interactions remain between MHCI and the peptide, at the minimum between the Cterminus of the peptide and the F-pocket of the MHCI antigen binding groove (31). Additionally, loss of ERAP1-peptide interactions may be substituted with ERAP1/MHCI interactions that could enhance peptide trimming rates.…”

“…We tested peptides in MHC complexes where both of the peptide termini are buried within the binding groove, peptides that have different length central bulges and peptides in MHCI complexes where the N-termini extends away from the groove at variable lengths. Our aim was, on the one hand, to test the generality of previous studies that suggested on-MHC trimming (23,31), and on the other hand, to test the relative kinetics of the two mechanisms, something that has not been addressed before. Kinetic analysis is critical to a detailed understanding of enzyme function, given that rates will determine whether a particular reaction is relevant in a biochemical pathway.…”

“…In contrast, MHCI have been shown to protect peptides from degradation by ERAP1 (30). Partial dissociation of the MHCI-bound peptide in conjunction with conformational rearrangements of ERAP1 towards more open states has been proposed as a mechanistic requirement to overcome these limitations, but direct experimental tests are lacking (27,31). Understanding the mode of ERAP1 peptide trimming is important because it alters our understanding of ERAP1's functional role in shaping the immunopeptidome in vivo.…”