Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation

Abstract

The cysteine protease legumain plays important functions in immunity and cancer at different cellular locations, some of which appeared conflicting with its proteolytic activity and stability. Here, we report crystal structures of legumain in the zymogenic and fully activated form in complex with different substrate analogs. We show that the eponymous asparagine-specific endopeptidase activity is electrostatically generated by pH shift. Completely unexpectedly, the structure points toward a hidden carboxypeptidase activity that develops upon proteolytic activation with the release of an activation peptide. These activation routes reconcile the enigmatic pH stability of legumain, e.g., lysosomal, nuclear, and extracellular activities with relevance in immunology and cancer. Substrate access and turnover is controlled by selective protonation of the S1 pocket (K M ) and the catalytic nucleophile (k cat ), respectively. The multibranched and context-dependent activation process of legumain illustrates how proteases can act not only as signal transducers but as decision makers.allostery | context-dependent activities | death domain | k cat -substrate specificity | electrostatic stability switch

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

“…While this manuscript was under preparation, Dall et al [32] reported the crystal structures of human pro-AEP and the active AEP in complex with the substrate analogs Z-Ala-Ala-AzaAsn-chloromethyl ketone (Z-AAN-CMK) and Ac-Tyr-Val-Ala-chloromethyl ketone (Ac-YVAD-CMK, an irreversible caspase-1 inhibitor). In their study, the pH-dependent activation was proposed, and an unexpected hidden asparagine (N)-specific carboxypeptidase activity was unveiled.…”

Proteolysis mediated by cysteine cathepsins and legumain—recent advances and cell biological challenges

“…Prolegumain, in clear contrast to most other endolysosomal proproteins, follows perhaps the most complicated and intriguing activation and inactivation pathways, which renders this cysteine peptidase one of the more interesting candidates to follow up on in the future (Dall and Brandstetter 2013;Ishii 1994;Kembhavi et al 1993;Watts et al 2005). The uncleaved full-length legumain, which does not share sequence homology with the cathepsins, is completely inactive.…”

Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment

“…Under physiological conditions, legumain is primarily expressed in kidney tubuli and contributes to renal tubular reabsorption 20,21 . Results of several studies have indicated that legumain expression is upregulated in a variety of different solid tumour types to a level that is positively correlated with the potential of malignancy [22][23][24] .…”