Universität zu Köln

Dall, Elfriede, Zauner, Florian B., Soh, Wai Tuck, Demir, Fatih ORCID: 0000-0002-5744-0205, Dahms, Sven O., Cabrele, Chiara, Huesgen, Pitter F. and Brandstetter, Hans ORCID: 0000-0002-6089-3045 (2020). Structural and functional studies ofArabidopsis thalianalegumain beta reveal isoform specific mechanisms of activation and substrate recognition. J. Biol. Chem., 295 (37). S. 13047 - 13065. ROCKVILLE: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. ISSN 1083-351X

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Abstract

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 ofArabidopsis thalianalegumain isoform beta (AtLEG beta) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform gamma (AtLEG gamma) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEG beta is concentration independent. Additionally, in AtLEG beta the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEG gamma, resulting in significantly different pH stability profiles. The crystal structure of AtLEG beta revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEG beta exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEG beta is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Dall, Elfriede UNSPECIFIED UNSPECIFIED UNSPECIFIED Zauner, Florian B. UNSPECIFIED UNSPECIFIED UNSPECIFIED Soh, Wai Tuck UNSPECIFIED UNSPECIFIED UNSPECIFIED Demir, Fatih UNSPECIFIED orcid.org/0000-0002-5744-0205 UNSPECIFIED Dahms, Sven O. UNSPECIFIED UNSPECIFIED UNSPECIFIED Cabrele, Chiara UNSPECIFIED UNSPECIFIED UNSPECIFIED Huesgen, Pitter F. UNSPECIFIED UNSPECIFIED UNSPECIFIED Brandstetter, Hans UNSPECIFIED orcid.org/0000-0002-6089-3045 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-319303
DOI:	10.1074/jbc.RA120.014478
Journal or Publication Title:	J. Biol. Chem.
Volume:	295
Number:	37
Page Range:	S. 13047 - 13065
Date:	2020
Publisher:	AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Place of Publication:	ROCKVILLE
ISSN:	1083-351X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language VACUOLAR PROCESSING ENZYME; ASPARAGINYL ENDOPEPTIDASE; PEPTIDE MACROCYCLIZATION; CIRCULAR PROTEINS; LEGUMAIN; SEED; ELECTROSTATICS; PURIFICATION; CYCLOTIDES; LIBRARIES Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/31930