Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

Abstract

Asparaginyl endopeptidase (AEP) is an endo/lysosomal cysteine endopeptidase with a preference for an asparagine residue at the P1 site and plays an important role in the maturation of toll-like receptors 3/7/9. AEP is known to undergo autoproteolytic maturation at acidic pH for catalytic activation. Here, we describe crystal structures of the AEP proenzyme and the mature forms of AEP. Structural comparisons between AEP and caspases revealed similarities in the composition of key residues and in the catalytic mechanism. Mutagenesis studies identified N44, R46, H150, E189, C191, S217/S218 and D233 as residues that are essential for the cleavage of the peptide substrate. During maturation, autoproteolytic cleavage of AEP's cap domain opens up access to the active site on the core domain. Unexpectedly, an intermediate autoproteolytic maturation stage was discovered at approximately pH 4.5 in which the partially activated AEP could be reversed back to its proenzyme form. This unique feature was confirmed by the crystal structure of AEP pH4.5 (AEP was matured at pH 4.5 and crystallized at pH 8.5), in which the broken peptide bonds were religated and the structure was transformed back to its proenzyme form. Additionally, the AEP inhibitor cystatin C could be digested by the fully activated AEP, but could not be digested by activated cathepsins. Thus, we demonstrate for the first time that cystatins may regulate the activity of AEP through substrate competition for the active site.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation

“…Biochemical experiments suggested that the enzymatic latency of plant legumain zymogens is conferred by the C-terminal pro-domain, which was reported to become (auto-)proteolytically cleaved at acidic pH (Kuroyanagi et al, 2002;Hiraiwa et al, 1999Hiraiwa et al, , 1997, in agreement with the crystal structures of mammalian legumains (Dall and Brandstetter, 2013;Zhao et al, 2014;Dall and Brandstetter, 2016;Li, 2014). It was reported that the catalytic-domain of legumain is instable at neutral pH (Dall and Brandstetter, 2013).…”

“…In mammalian legumain, the electrostatically anchored LSAM domain is released from the protease domain upon protonation of the peptidase-harboured Asp and Glu side chains (Dall and Brandstetter, 2013) , (Zhao et al, 2014). By contrast, we found a predominantly hydrophobic protease-LSAM interface (yellow color code in Fig.…”

“…4). This unique, plant-specific α6-LSAM module accounts for the increased pH-stability at neutral pH and the significantly stronger interaction in AtLEGγ rather than in the mammalian legumains, although an intermediate co-migration of human and mouse peptidase domain with LSAM at pH>4.5 was reported (Zhao et al, 2014;Dall and Brandstetter, 2013). For a direct (α6- (Fig.…”

“…Ligase activity has recently also been reported in mammalian legumain (Dall et al, 2015b) , (Zhao et al, 2014). However, in vitro proof for ligase activity of Arabidopsis thaliana legumain isoforms is lacking.…”

Overexpression of asparaginyl endopeptidase is significant for esophageal carcinoma metastasis and predicts poor patient prognosis

“…AEP, currently the only known asparaginyl endopeptidase in the mammalian genome, is a member of the C13 family in the MEROPS database classification of peptidases, whereas all other lysosomal cysteine proteases identified to date are grouped in the C1 family (6,7). The strict specificity of AEP to asparagine bonds is notable (8). AEP has been demonstrated to contribute important functions in kidney physiology, immunity, atherogenesis and bone metabolism (9)(10)(11)(12)(13)(14)(15).…”