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.2011 May 10;108(19):7745-50.
doi: 10.1073/pnas.1101262108. Epub 2011 Apr 20.

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

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

Grazyna Kochan et al. Proc Natl Acad Sci U S A..

Abstract

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)(18)-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K(528)R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overall structure and different conformational states of human ERAP1. (A) Closed form of ERAP1. ERAP1 consists of four domains (I: green, II: orange, III: yellow, IV: teal). The first crystal form showed one molecule where the active site of domain II is shielded by domain IV. N-glycosylation sites are colored in magenta. (B) Open form of ERAP1. A second crystal form contained three molecules of ERAP1 and revealed that it is indeed a highly dynamic molecule. Domain IV of molecule 2 moves away from the protease domain (II) and permits substrate access to the active site. Note that parts of domain IV, facing toward the catalytic domain, were disordered. (C) A distance plot shows the Cα-Cα distance of equivalent residues of the closed and the three open molecules. Conformational changes are mostly confined to domain III and IV. Regions that were not defined by electron density in the open molecules are blank. The insets show superpositions of the closed state (gray) and each open molecule. The different rotary motion for the individual molecules is indicated by arrows.
Fig. 2.
Fig. 2.
Active site and internal cavity of ERAP1. (A) Bestatin binding to the active site of ERAP1. Bestatin is colored in orange and the surrounding electron density is anFo-Fc map calculated in the absence of the molecule and drawn at 3.0σ. (B) Superposition of open (gray) and closed (colored) state of ERAP1. The movement of Tyr438 is highlighted and Phe433, which forms part of the S1 specificity pocket in the active protease is disordered in the open state. (C) Putative C-terminal substrate binding site within the internal cavity of ERAP1.
Fig. 3.
Fig. 3.
Altered N-terminal peptide trimming by the AS associated ERAP1 K528R mutant. (A) Extracted ion chromatograms (EIC) of the peptide cleavage product TANRELIQQEL after trimming of QITANRELIQQEL by ERAP1 wild type and the K528R mutant. Peptide precursor and enzymes were incubated for different sampling times revealing differential kinetic activity between both variants. (B) Relative quantitation of the EICs for the substrate peptide QITANRELIQQEL shows higher activity of the ERAP1 wild type. The intermediate product TANRELIQQEL is further processed by ERAP1 wild type while the K528R variant stops processing this peptide at the 11-mer stage. (C) Relative quantitation of the EICs for all observed peptide intermediates. While the wild type reaches equilibrium at the 8- and 9-mer, the K528R variant stops processing the 11-mer.
Fig. 4.
Fig. 4.
Polymorphism associated with ankylosing spondylitis. (A) Surface representation of ERAP1 Mutations associated to AS are colored in red. Localization of Met349Val in the substrate pocket is only visible in the open conformation. (B andC) Interactions of Lys528 with surrounding amino acids in the closed state (B) and the open state (C). The side chain of Asn414 was disordered in the open state, as were interactions with domain IV. Parts of the surface carbohydrate moiety (NAG1414) attached to Asn414 is depicted in the closed form (B).
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