Structural insights into the molecular ruler mechanism of the endoplasmic reticulum aminopeptidase ERAP1

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an essential component of the immune system, because it trims peptide precursors and generates the N--restricted epitopes. To examine ERAP1's unique properties of length- and sequence-dependent processing of antigen precursors, we report a 2.3 Å resolution complex structure of the ERAP1 regulatory domain. Our study reveals a binding conformation of ERAP1 to the carboxyl terminus of a peptide, and thus provides direct evidence for the molecular ruler mechanism.

Effects of polymorphic variation on the mechanism of Endoplasmic Reticulum Aminopeptidase 1

“…We focused our analysis on two key ERAP1 SNPs, located at positions 528 and 730, since they are the two most commonly discovered SNPs in genetic studies and they represent typical examples of SNP locations: one within the internal cavity of ERAP1 and one on the outside. The polymorphic position 730 lies within the extended internal cavity of ERAP1 and although it is 30 Å away from the active site, it may still interact with the C-terminal moiety of a long peptide substrate as previously reported based on crystallographic data (Gandhi et al, 2011). This observation can potentially explain our experimental results: the nature of amino acid at that location may affect recognition of the longer peptide substrates and therefore length selection.…”

“…Position 730 lies within the internal cavity of ERAP1 that has been hypothesized to accommodate the peptidic substrates and is in close proximity to a proposed ERAP1 regulatory region (Fig. S2) (Gandhi et al, 2011). It is therefore, possible that SNP at position 730 interferes directly with the ability of longer peptides to bind or to self-activate their own trimming as previously proposed (Nguyen et al, 2011).…”

“…Therefore, the open state has been suggested to facilitate initial substrate capture and the closed state to enhance catalysis by organizing catalytic residues and specificity pockets. Larger peptides may facilitate this transition through a mechanism of self-activation involving a still unmapped regulatory site in the enzyme (Nguyen et al, 2011;Gandhi et al, 2011;Kochan et al, 2011). Overall, it appears that ERAP1 has evolved to specialize for generating antigenic peptides for MHCI.…”

Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing

“…The weaker density toward the C terminus of the peptide, the lack of deep binding pockets, and the molecular dynamics simulations all corroborate the idea that the C-terminal end of the peptide is not specifically recognized by IRAP and that the structure is the result of opportunistic interactions and shape complementarity in the limited space in between domains II and IV. This is in sharp contrast to what has been proposed for the homologous ERAP1, for which a specific recognition of the C terminus of the peptide-ligand has been proposed based on both functional and structural data (39,42,43). To further investigate this, we tested whether IRAP has some of the enzymatic properties that are unique to ERAP1 and have been associated to recognition of the C terminus of the peptide.…”

Crystal structure of a polypeptide’s C-terminus in complex with the regulatory domain of ER aminopeptidase 1

“…However, the actual mechanism of ERAP1 sequence and length-dependent activity is elusive since there is no structural information available for ERAP1-peptide complex. To analyze ERAP1's unique peptide recognition and substrate selection, we previously reported a 2.3Å resolution structure of the ERAP1 regulatory (ERAP1_R) domain forming a complex with a His 6 peptide 20 . Based on that complex structure and supporting activation assays using various His-containing peptides, we proposed a modular organization of ERAP1 for its molecular ruler mechanism 20 .…”