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.
To determine the location of the proteinase in the covalent serpin-proteinase complex we prepared seven single-cysteine-containing variants of the Pittsburgh variant of the serpin ␣ 1 -proteinase inhibitor, and we labeled each cysteine with the dansyl f luorophore. The dansyl probes were used to determine proximity of the proteinase trypsin in covalent and noncovalent complexes with the serpin, both by direct perturbation and by f luorescence energy transfer from tryptophans in trypsin to dansyl. Large direct effects on dansyl f luorophores were seen for only two positions in covalent complex and one position in noncovalent complex. Distances ranging from <14 Å to 64 Å were used to severely constrain possible structures for the complex. The structure consistent with both distance constraints and direct perturbations of the dansyl f luorophores placed the proteinase at the distal end of the serpin from the initial docking site. This position for the proteinase requires complete translocation of the proteinase from one end of the serpin to the other and full insertion of the reactive center loop into -sheet A to form the kinetically trapped complex. The consequent tight juxtapositioning of serpin and proteinase could explain how distortion of the proteinase active site can occur and hence how many combinations of serpin and proteinase can be inhibited by a common conformational change mechanism.Elucidation of the structure of the serpin-proteinase complex remains the holy grail in trying to understand how serpins inhibit serine proteinases by a kinetic trap mechanism. Serpins inhibit proteinases by a branched pathway, suicide substrate inhibition mechanism (Fig. 1) in which a peptide bond in the exposed reactive center loop is initially recognized as an appropriate proteolytic cleavage site by proteinase, which thereby forms an initial noncovalent Michaelis complex. Formation of this complex is followed by attack by the proteinase active site on the peptide bond (1). Although there may be special cases of particular serpin-proteinase pairs where reaction stops at the Michaelis complex (2), formation of the serpin-proteinase complex under most circumstances involves progression of this initial noncovalent complex to the covalent acyl enzyme intermediate (E-I in Fig. 1), release of the newly formed amino terminus (P1Ј residue) (3), and insertion of the now unconstrained reactive center loop into -sheet A. Because the proteinase is covalently linked to the P1 residue of the serpin through an ester linkage, any insertion of the reactive center loop must involve concomitant proteinase translocation. At a point during insertion of the reactive center loop into -sheet A, a physical interaction is thought to occur between the serpin and the proteinase that is sufficiently large enough to alter the properties of the proteinase (4) and thereby render it catalytically incompetent. The resulting structure represents the kinetically trapped covalent serpin-proteinase complex (E-I † ).Several studies support such a g...
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.
Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. molecular structure | adaptive immunity | major histocompatibility molecules | specificity | kinetics
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.
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