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

Abstract

The vacuolar cysteine protease legumain can cleave and selectively rebuild peptide bonds, thereby vastly expanding the sequential repertoire of biomolecules. In this context, plant legumains have recently attracted particular interest. Furthermore, legumains have important roles in many physiological processes, including programmed cell death. Their efficient peptide bond ligase activity has gained tremendous interest in the design of cyclic peptides for drug design. However, the mechanistic understanding of these dual activities is incomplete and partly conflicting. Here we present the crystal structure of a plant legumain, Arabidopsis thaliana isoform-γ (AtLEGγ). Employing a conserved legumain fold, the plant legumain AtLEGγ revealed unique mechanisms of auto-activation, including a plant-specific two-chain activation state, which remains conformationally stable at neutral pH, which is a prerequisite for full ligase activity and survival in different cell compartments. The charge distribution around the α6-helix mediates the pH-dependent dimerization and serves as a gatekeeper for the active site, thus regulating its protease and ligase activity.

The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity

“…The crystal structure of the pro‐enzyme form of butelase 1 is strikingly similar to those of other AEPs, where an unstructured linker region between the core domain and the cap could provide the flexibility required for self‐cleavage of the cap domain upon activation. Furthermore, the crystal structure lends further support for the cap domain forming a dimer interaction surface, as suggested previously (Yang et al ., ; Zauner et al ., ). Notably, we have chosen to model a SNN residue adjacent to the catalytic Cys despite the crystal structure being the pro‐form of butelase 1.…”

Structural determinants for peptide-bond formation by asparaginyl ligases

“…AtLEGγ (Arabidopsis thaliana) (45) catalyze both ligation and hydrolysis products from peptide substrates carrying AEP-recognition signals at near-neutral pH (6-7.5). Other AEPs, such as butelase 2, OaAEP2, and HaAEP1 (sunflower Helianthus annuus), display predominantly protease activity even at neutral pH, with a very low level of ligase activity (35,45,46). In contrast to these "bifunctional" or "predominant" AEPs, butelase 1 and OaAEP1b catalyze the formation of ligation products essentially devoid of any hydrolytic product at near-neutral pH, and their ligase activity is preponderant even under mild acidic conditions (pH < 6).…”

“…What are the molecular mechanisms differentiating AEPs and PALs? Despite the publication of several plant AEP crystal structures, including both proenzymes and active forms (34,(45)(46)(47), the structural determinants that underpin their nature as protease or ligase are still unresolved. Enzymes from both extremes share high structural similarities (e.g., OaAEP1b and HaAEP1).…”

The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

“…The crystal structure of the proenzyme form of butelase 1 bears a strikingly similar structure to those of other AEPs, where an unstructured linker region between the core domain and the cap could provide the flexibility required for self-cleavage of the cap domain upon activation. Furthermore, the crystal structure lends further support for the cap domain forming a dimer interaction surface as suggested previously (Yang et al, 2017;Zauner et al, 2018b). Notably, we have chosen to model a succinimide residue adjacent to the catalytic Cys despite the crystal structure being the pro-form of butelase 1.…”