Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2

Abstract

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the `molecular-ruler' mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure-function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.

Figure 1

4–12% SDS–PAGE under reducing conditions showing the purified ERAP2_50–­960 running at approximately 95 kDa next to SeeBlue Plus 2 molecular-weight markers from Invitrogen (the orange marker is running at approximately 98 kDa).

Figure 2

Crystals of ERAP2_50–960 grown by vapour diffusion at 295 K in a mother liquor consisting of 0.5 M MES pH 7.0, 0.2 M KSCN, 0.02 M CaCl₂, 12%(w/v) PEG monomethylether 5000. The crystal in the centre of the picture is approximately 0.03 × 0.1 × 0.1 mm in size.

Figure 3

X-ray diffraction pattern recorded in-house from an ERAP2_50–960 crystal (0.5° oscillation) with visible diffraction to 3.3 Å resolution.