The ERAP1 active site cannot productively access the N-terminus of antigenic peptide

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed “ontο MHCI” trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1.

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Fig: Construction and validation of a disulfide-linked HLA-B*08/peptide complex. Panel (A), molecular model of the HLA-B*08(E76C)-ARAALRSRYWCI complex (peptide shown in green sticks, disulfide bond between the HLA residue Cys76 and peptide residue Cys11, is indicated). Panel (B), size-exclusion chromatogram after refolding of HLA-B*08(E76C) with the peptide ARAALRSRYWCI. Panel (C), SDS-PAGE of peaks A and B of chromatogram shown in Panel (B). Panel (D), MALDI-TOF–MS of purified complex in the presence of 100 mM DTT. Panel (E), MALDI-TOF–MS of purified complex in the absence of DTT. Panel (F), 1st derivative of thermal shift assay of purified complex (three repetitions shown). Panel (G), kinetic analysis of binding of SYPRO Οrange to purified complex HLA-B*08(E76C)-ARAALRSRYWCI (each data point is the average of three repetitions). Panel (H) Kinetic analysis of binding of SYPRO Οrange to non-covalent complex HLA-B*08 – ARAALRSRYWAI (each data point is the average of two repetitions).

Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

Mavridis, G., Mpakali, A., Zoidakis, J. et al. The ERAP1 active site cannot productively access the N-terminus of antigenic peptide precursors stably bound onto MHC class I. Sci Rep 11, 16475 (2021). https://doi.org/10.1038/s41598-021-95786-x

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