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.
Legumain is a dual-function protease–peptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana legumain isoform γ (AtLEGγ) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEGγ structures disclosed a substrate-induced disorder–order transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEGγ ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substrate–like binding to the active site–binding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2′–S2′, to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEGγ active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.
The cysteine protease enzyme legumain hydrolyzes peptide bonds
with high specificity after asparagine and under more acidic conditions
after aspartic acid [BakerE. N.BakerE. N.7003158J. Mol. Biol.1980141441484; BakerE. N.BakerE. N.859183J. Mol. Biol.1977111207210; DrenthJ.DrenthJ.952885Biochemistry19761537313738; MenardR.MenardR.J. Cell. Biochem.1994137; PolgarL.PolgarL.689035Eur. J. Biochem.197888513521; StorerA. C.StorerA. C.7845227Methods Enzymol.1994244486500. Remarkably,
legumain additionally exhibits ligase activity that prevails at pH
> 5.5. The atomic reaction mechanisms including their pH dependence
are only partly understood. Here we present a density functional theory
(DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of
the detailed reaction mechanism of both activities for human legumain
in solution. Contrasting the situation in other papain-like proteases,
our calculations reveal that the active site Cys189 must be present
in the protonated state for a productive nucleophilic attack and simultaneous
rupture of the scissile peptide bond, consistent with the experimental
pH profile of legumain-catalyzed cleavages. The resulting thioester
intermediate (INT1) is converted by water attack on the thioester
into a second intermediate, a diol (INT2), which is released by proton
abstraction by Cys189. Surprisingly, we found that ligation is not
the exact reverse of the proteolysis but can proceed via two distinct
routes. Whereas the transpeptidation route involves aminolysis of
the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted
ligation route was found. Given legumain’s important roles
in immunity, cancer, and neurodegenerative diseases, our findings
open up possibilities for targeted drug design in these fields.
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.
We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.
Wirp räsentieren eine neue Methode zur Proteinmarkierung,d ie auf der kovalenten enzymatischen Phosphocholinierung einer spezifischen Octapeptidsequenz intakter Proteine beruht. Das Enzym AnkX aus Legionellen wurde etabliert, um funktionalisierte Phosphocholingruppen aus synthetischen CDP-Cholin-Derivaten auf N-und C-Termini, sowie interne Schleifenregionen zu übertragen. Zudem kann die kovalente Modifikation durchd as Legionellen-Enzym Lem3 hydrolytische ntfernt werden. Lediglich eine 8Aminosäuren kurze Peptidsequenz und eine kleine Verbindungsgruppe (PEG-Phosphocholin) sind für die effiziente Proteinmarkierung erforderlich.
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