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

Florian B Zauner,Elfriede Dall,Chiara Cabrele,Christof Regl,Hans Brandstetter,Christian G Huber,Luigi Grassi

doi:10.1105/tpc.17.00963

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 autoactivation, 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.

Highlights

In contrast to mammals with only one isoform, plants encode at least four functional isoforms of the cysteine protease legumain (C13 family, EC 3.4.22.34)
The catalytic domain is covered by two short antiparallel b-sheets, serving as primed and nonprimed substrate recognition sites in the mature asparaginyl endopeptidases (AEPs) form and as an anchoring interface to the C-terminal LSAM domain in the zymogen
We identified an independently evolved and mechanistically drastically different proteolytic autoactivation compared with mammalian legumain, with a stable two-chain intermediate (Figure 3; Supplemental Figure 4); the latter can be further activated to the classical asparaginyl endopeptidase (AEP) only under reducing conditions

Summary

Introduction

In contrast to mammals with only one isoform, plants encode at least four functional isoforms of the cysteine protease legumain (C13 family, EC 3.4.22.34). The vegetative type legumains, like Arabidopsis isoform g, have critical roles in plant programmed cell death (reviewed in Hatsugai et al, 2015) and partially substitute the orthologous caspases, which are absent in plants (Bonneau et al, 2008). AtLEGg exhibits caspase-1 like activity and its expression in Besides proteolysis, certain plant legumains are known to be efficient peptide ligases and cyclases (Conlan et al, 2010; Craik and Malik, 2013; Nguyen et al, 2014, 2016; Bernath-Levin et al, 2015a; Harris et al, 2015; Yang et al, 2017; Saska et al, 2007). In vitro proof for ligase activity of Arabidopsis legumain isoforms is lacking

Methods

Results

Conclusion