The Internal Sequence of the Peptide-Substrate Determines Its N-Terminus Trimming by ERAP1

Abstract

BackgroundEndoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.Methodology/Principal FindingsIn an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains.Conclusions/SignificanceTo our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

“…Domain I (residues 46-254) of ERAP1 (Fig. 1A) is composed of a central, saddleshaped eight-stranded β-sheet (β1, 2, 4, 5, 8 and β11, 13,14). This central motif is on one end flanked by a three-stranded β-sheet (β3, 6, 7) and on the opposite end by a four-stranded β-sheet (β9 þ 10 and β12 þ 15) that packs against the catalytic domain and interacts with domain IV via an elongated loop connecting strands 9 and 10.…”

HLA-B27, but Not HLA-B7, Immunodominance to Influenza Is ERAP Dependent

“…Our peptide immunization studies suggest that the NP383 epitope is most likely generated as an N-terminally extended 14-mer that is subsequently trimmed in the ER by ERAP before being loaded into the MHC I peptide-binding groove. ERAP has been shown to cleave polypeptides at specific sites expressing leucine (L), methionine (M), phenylalanine (F), and tyrosine (Y) in the amino acid sequence (25,26). The natural 14-mer sequence of NP383-391 (i.e., TLELRSRYWAIRTR) contains two leucines upstream of NP383-391 at positions 11 and 13, which provide potential cleavage sites for ERAP.…”

The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis

“…Through a mechanism named 'molecular ruler', whereby the enzyme itself acts as a peptide-length template, ERAP1 trims peptides of 9-16 residues very efficiently while sparing shorter peptides [66][67][68]. The substrate specificity of ERAP1 is dictated by the N-and C-terminal residues of the peptide as well as by the internal sequence [67,[69][70][71]. ERAP1 shows preferences for hydrophobic residues, while basic and acidic amino acids are poor substrates; finally, Pro is never hydrolyzed [69,70].…”