Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here we present the crystal structure of Arabidopsis thaliana legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. While the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions as in AtLEGγ, resulting in significantly different pH-stability profiles. The crystal structure of AtLEGβ reveiled unrestricted non-prime substrate binding pockets, consistent with the broad substrate specificity as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. While cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform specific differences will allow to identify and rationally design efficient ligases with application in biotechnology and drug development.

Keywords: crystal structure; cysteine protease; pH regulation; plant biochemistry; protein stability; structural biology; transpeptidation.