Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition

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 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.

Plant asparaginyl endopeptidases and their structural determinants of function

“…AEPs are synthesised in the inactive form consisting of the N-terminal 'core' domain, which contains the active site, and a smaller C-terminal auto-inhibitory 'cap' domain, which is also termed the Legumain Stabilisation and Activity Modulation domain as it blocks access to the active site, conferring enzymatic latency and protein stability at neutral pH conditions (Figure 2) [80]. Conversion of AEP into the active form has been studied in exquisite detail in two isoforms of A. thaliana AEP (AtAEP2 and AtAEP3) [80][81][82]. AEP activation occurs via autocatalytic cleavage around a flexible linker region between the cap and core domains, and requires disruption of electrostatic interactions between the two domains under acidic conditions [80][81][82].…”

“…Conversion of AEP into the active form has been studied in exquisite detail in two isoforms of A. thaliana AEP (AtAEP2 and AtAEP3) [80][81][82]. AEP activation occurs via autocatalytic cleavage around a flexible linker region between the cap and core domains, and requires disruption of electrostatic interactions between the two domains under acidic conditions [80][81][82].…”

“…AEP residues that interact with substrate residues preceding the scissile peptide bond, have been shown to influence transpeptidation efficiency. Substrate affinity, hypothesised by some to positively influence transpeptidase activity, was attributed to the non-primed region [ 81 ]. However, the complex interplay amongst the residues in this region makes it difficult to rationalise mechanistic explanations for their influence on AEP activity.…”

“…AEPs such as HaAEP1 that have been structurally and biochemically characterised as predominant peptidases have turned out to also have high cyclic to linear product ratio when assayed with different substrates [ 14 ]. Asparagine/aspartate preference is pH-dependent — aspartate was hypothesised to be accepted as a P1 residue below pH 5.5 because protonating the aspartate carboxylic sidechain makes it sterically and electrostatically similar to asparagine, which is the preferred P1 residue at higher pH conditions [ 3 , 81 , 91 ]. Studies on AtAEP3 showed that transpeptidation activity is typically confined to near-neutral pH conditions [ 80 ] where N-terminal amino groups are more likely deprotonated, either on their own or by a deprotonated catalytic histidine, and therefore able to perform nucleophilic attack on the acyl-enzyme intermediate [ 11 ].…”

MANTI: Automated Annotation of Protein N-Termini for Rapid Interpretation of N-Terminome Data Sets

“…MANTI has already been successfully used for a multitude of different samples, including N-terminome data sets from both animals 5 , 29 and plants. 16 , 20 , 30 , 31 …”