Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

ABSTRACT Legumains 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 k cat -tuning switch embedded within the substrate, and highlighted its potential for developing high-performance fluorogenic substrates. Collectively, these findings advance the mechanistic understanding of legumain’s transpeptidase activity and provide a framework for developing selective probes and inhibitors across the legumain family and related cysteine proteases. GRAPHICAL ABSTRACT