Klaushofer, R. | bioRxiv Science

bioRxiv2025

Positional Scanning and Computational Modeling Reveal Determinants of Legumain Transpeptidase Activity

ABSTRACTLegumains are cysteine proteases that, in addition to their canonical hydrolase function, can act as peptide ligases or transpeptidases. In humans, this activity becomes particularly relevant under pathophysiological conditions, where legumain relocalizes to near-neutral pH compartments favoring ligation/transpeptidation over hydrolysis. Here, we combined in vitro positional scanning with in silico substrate profiling to elucidate the substrate determinants governing human legumain-mediated peptide cyclization. We identified glycine residues at P1'' and P1' and basic residues at P2'/P2'' as key determinants of human legumain-mediated peptide cyclization. Guided by these insights, we designed an optimized substrate exhibiting substantially enhanced cyclization efficiency. Computational analysis not only recapitulated the experimental observations but also predicted a covalent inhibition mechanism involving a P1' cysteine, revealed a kcat-tuning switch embedded within the substrate, and highlighted its potential for developing high-performance fluorogenic substrates. Collectively, these findings advance the mechanistic understanding of legumains transpeptidase activity and provide a framework for developing selective probes and inhibitors across the legumain family and related cysteine proteases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/693912v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@121ffcborg.highwire.dtl.DTLVardef@120a587org.highwire.dtl.DTLVardef@536fc7org.highwire.dtl.DTLVardef@1cfaa67_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Conformational and Functional Regulation of SET by Legumain Cleavage

The cysteine protease legumain typically localizes to the endolysosomal system, where it is an important player in the immune system. However, in the context of Alzheimers disease (AD), legumain has been shown to be translocated to the cytosol, where it cleaves SET, synonymously termed TAF-1 or I2PP2A, an inhibitor of protein phosphatase 2A. SET is primarily found in the nucleus, where it regulates gene transcription, cell cycle progression, and histone acetylation, but can also translocate to the cytoplasm where it regulates cell migration and is implicated in neuronal apoptosis in AD. In this study, we demonstrate that legumain cleaves SET at two major sites: Asn16 at the N-terminal end and Asn175 at the earmuff domain. Contrary to previous findings, our biochemical and crystallographic experiments reveal that the corresponding N- and C-terminal cleavage products remain bound in a stable complex, rather than dissociating. Additionally, we show that the C-terminal acidic stretch of SET is essential for its binding to histone 1, and that cleavage impairs this interaction. Finally, we demonstrate that SET positively modulates PP2A activity. This effect is however abolished upon cleavage by legumain.

molecular biology↗