Structural analyses of Arabidopsis thaliana legumain γ reveal differential recognition and processing of proteolysis and ligation substrates

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

“…In the absence of differences between active sites, it is likely that differences in the substrate binding pockets are what define substrate specificity and catalytic efficiency of AEPs, as has been suggested for the improved ligation efficiency of butelase 1 compared to OaAEP1 (Yang et al, 2017). In comparison to previously published crystal structures, butelase 1 exhibits an extended, flexible α5-β6 loop that might become more rigid upon substrate binding and play a role in guiding the N-terminus of a substrate for attack and resolution of a thioacyl intermediate, resulting in peptide cyclization as recently suggested for A. thaliana AEP3 (Zauner et al, 2018a). This loop has also recently been highlighted as a 'marker of ligase activity' with an extended loop proposed to be absent in ligases, and although butelase 1 does not exhibit such a deletion it was shown to display a more hydrophobic loop than its cleavage-favoring counterpart butelase 2 (Jackson et al, 2018).…”

“…The catalytic residues in the active site of butelase 1, Asn59, His165, and Cys207, align closely with the catalytic residues of OaAEP1 (Asn70, His175, and Cys217), HaAEP1 (Asn73, His178, and Cys220), and AtAEP3 (Asn72, His177, and Cys219). Furthermore, we chose to model a succinimide (SNN) residue at position 164 due to the preponderance of this aspartimide at this position (N-terminally adjacent to the catalytic His165) in other AEP crystal structures (Dall et al, 2015;Haywood et al, 2018;Zauner et al, 2018a), along with an associated reduction in steric clashes when compared with the modelled alternative Asp164 residue (Supplemental Figure 4). In particular, the short 1.9 Å distance between the OH group of Tyr162 and an Oδ from the side chain of Asp164, which is extended to 2.9 Å with the O5 of SNN, was notable in guiding our decision to model residue 164 as SNN.…”

“…Mutating a bulky Cys residue located in the substrate channel to a smaller Ala residue greatly increased the rates of macrocyclization activity of an AEP from Oldenlandia affinis (Yang et al, 2017). Furthermore, recent sequence analysis and molecular modeling has revealed a range of residues that together contribute to ligation efficiency (Jackson et al, 2018;James et al, 2018;Zauner et al, 2018a).…”

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

“…VPEs are endopeptidases belonging to a different clan of cysteine proteases which are not sensitive to E‐64 and display a strict specificity for cleavage after asparagine or aspartic acid residues . Such cleavage events were observed in the case of PG9 and 2G12‐I19R processing by apoplastic proteases, and these reactions were partially sensitive to the known VPE inactivators Ac‐YVAD‐CMK and Z‐VAD‐FMK .…”

“…VPEs are endopeptidases belonging to a different clan of cysteine proteases which are not sensitive to E-64 and display a strict specificity for cleavage after asparagine or aspartic acid residues. [46] Such cleavage events were observed in the case of PG9 and 2G12-I19R processing by apoplastic proteases, and these reactions were partially sensitive to the known VPE inactivators Ac-YVAD-CMK and Z-VAD-FMK. [47] However, PG9 degradation by apoplastic fluid was not affected by the addition of human cystatin C (Puchol Tarazona, unpublished observation), whose chicken homologue is a potent VPE inhibitor.…”