5OBT

Fully activated A. thaliana legumain isoform gamma in complex with Ac-YVAD-CMK

Summary for 5OBT

• Entry DOI: 10.2210/pdb5obt/pdb
• Related PRD ID: PRD_002086
• Descriptor: Vacuolar-processing enzyme gamma-isozyme, Ac-YVAD-CMK, ... (4 entities in total)
• Functional Keywords: legumain, asparaginyl endopeptidase, vacuolar processing enzyme, peptide cyclase, hydrolase
• Biological source: Arabidopsis thaliana (Thale cress); More
• Total number of polymer chains: 6
• Total formula weight: 66558.38

Authors

Zauner, B.F.,Dall, E.,Brandstetter, H. (deposition date: 2017-06-29, release date: 2018-04-11, Last modification date: 2024-11-06)

Primary citation

Zauner, F.B.,Elsasser, B.,Dall, E.,Cabrele, C.,Brandstetter, H.
Structural analyses ofArabidopsis thalianalegumain gamma reveal differential recognition and processing of proteolysis and ligation substrates.
J. Biol. Chem., 293:8934-8946, 2018

PubMed Abstract: 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 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 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 cyclization. -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.

PubMed: 29628443
DOI: 10.1074/jbc.M117.817031

Experimental method

X-RAY DIFFRACTION (1.5 Å)