ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

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.

The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8 + T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-regulates ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to reduced susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings reveal a novel viral miRNA-based CTL evasion mechanism that targets a key step in the MHC class I antigen-processing pathway.MHC class I molecule binds peptides and presents them on the cell surface for recognition by CD8 + T cells. Cytosolic peptides generated by the proteasomes and prematurely translated peptides are transported to the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) complex 1, 2 . Some of these peptides are further processed by ER-resident aminopeptidases, such as ERAP1, and peptide trimming by ERAP1 (also known as A-LAP, ARTS-1, and PILS-AP) in the ER is a crucial step for determining the quality and quantity of optimal antigenic peptide production and the stability of the MHC class I-β 2 m-peptide heterotrimer [3][4][5][6] . ERAP1 trims relatively long * To whom correspondence should be addressed: Department of Biological Sciences, Seoul National University, Seoul 151-747, Korea, (Phone) +822-880-9233 (Fax) +822-872-1993 S.R.R cloned the HCMV-specific CTLs. S.K and K.A designed the overall study and wrote the paper. NIH Public AccessAuthor Manuscript Nat Immunol. Author manuscript; available in PMC 2012 December 20. peptides efficiently in a sequence-specific manner, resulting in the accumulation of 8-9 amino acid-long optimal peptides 5,7,8 . ERAP1 therefore acts as a 'molecular ruler' for antigenic peptide production 9 . In addition, genome-wide association studies have associated nonsynonymous single nucleotide polymorphisms in ERAP1 with ankylosing spondylitis 4 . Additionally, ERAP1 has non-peptide processing functions via its role in shedding of cytokine receptors 4 .MiRNAs are small RNAs 19 to 23 nucleotides long that regulate gene expression by complete or partial base-pairing with the 3′-untranslated region (UTR) of their target mRNA which leads to mRNA cleavage, destabilization, or translational repression 10 . Since the first report in 2004 that viruses express miRNAs 11 , numerous viral miRNAs have been discovered and are mainly related to viral proliferation and survival-related immune evasion, although this is based on a limited number of studies 12 . The β-herpesvirus HCMV expresses at least 14 miRNAs during productive infection 13,14 . One HCMV-encoded miRNA, miR-UL112-1 targets 3 HCMV genes involved in viral r...

Synopsis ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and Insulin Regulated aminopeptidase (IRAP) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding onto MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorogenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the primary specificity (S1) pocket. Molecular modeling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP however, does not achieve this dual specificity by simply combining structural features of ERAP1 and 2, but rather by a unique amino acid change at position 541. Our results provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.

ER aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered antigen processing by these enzymes is a causal link to disease etiology but the molecular mechanisms are obscure. We report here that the common ERAP2 SNP rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity-switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans.

The processing of MHC class I antigenic precursor peptides by the endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 is an important event in the cell biology of antigen presentation. To date, the molecular context by which the ERAP enzymes trim precursor peptides, and how ERAPs shape peptide repertoires, remain open questions. Using ERAP1 and ERAP2 heterodimers (ERAP1/2), and N-terminally extended model and natural peptides in their free and HLA-B*0801-bound forms, we characterized the mode of action of ERAPs. We provide evidence that ERAP1/2 can trim MHC I-bound precursor peptides to their correct and final lengths, albeit more slowly than the corresponding free precursors. Trimming of MHC I-bound precursors by ERAP1/2 increases the conformational stability of MHC I/peptide complexes. From the data, we propose a molecular mechanistic model of ERAP1/2 as peptide editors. Overall, our study provides new findings on a significant issue of the ERAP-mediated processing pathway of MHC class I antigens.

ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in the immune system by trimming peptides for loading onto major histocompatibility complex proteins. Here, we report discovery of the first inhibitors selective for ERAP1 over its paralogues ERAP2 and IRAP. Compound 1 (N-(N-(2-(1H-indol-3-yl)­ethyl)­carbamimidoyl)-2,5-difluorobenzenesulfonamide) and compound 2 (1-(1-(4-acetylpiperazine-1-carbonyl)­cyclohexyl)-3-(p-tolyl)­urea) are competitive inhibitors of ERAP1 aminopeptidase activity. Compound 3 (4-methoxy-3-(N-(2-(piperidin-1-yl)-5-(trifluoromethyl)­phenyl)­sulfamoyl)­benzoic acid) allosterically activates ERAP1’s hydrolysis of fluorogenic and chromogenic amino acid substrates but competitively inhibits its activity toward a nonamer peptide representative of physiological substrates. Compounds 2 and 3 inhibit antigen presentation in a cellular assay. Compound 3 displays higher potency for an ERAP1 variant associated with increased risk of autoimmune disease. These inhibitors provide mechanistic insights into the determinants of specificity for ERAP1, ERAP2, and IRAP and offer a new therapeutic approach of specifically inhibiting ERAP1 activity in vivo.