The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity

Abstract

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

Structural basis for a natural circular permutation in proteins

“…We modelled a succinimide intermediate (SNN) at Asp157 to obtain a better fit with the electron density at this position (Supplemental Figure 9A). The presence of SNN is consistent with all other plant structures except OaAEP1 (Yang et al, 2017;Haywood et al, 2018;Zauner et al, 2018b;Hemu et al, 2019;James et al, 2019), although OaAEP1 does have electron density suggesting that succinimide may in fact be present (James et al, 2019). Gln335, which is part of the cap domain, occupies the S1 pocket of the active site in the zymogen (according to nomenclature by Schechter and Berger where P1, P2 and so on refer to residues N-terminal to the cleavage site, whereas P1′, P2′ and so on are C-terminal to it, and where the corresponding binding sites on the protease are termed S2, S1, S1′, S2′, etc.)…”

“…CeAEP1 is structurally similar to previously solved plant AEP structures: Helianthus annuus AEP1 or HaAEP1 6AZT (Haywood et al, 2018), AtLegγ 5NIJ (Zauner et al, 2018b), Oldenlandia affinis AEP1 or OaAEP1 5H0I (Yang et al, 2017), Viola yedoensis peptide asparaginyl ligase 2 or VyPAL2 6IDV (Hemu et al, 2019), and butelase1 6DHI (James et al, 2019) with an r. m. s. d. of 1.0-1.1 Å over 400-430 Cαatoms between CeAEP1 and the other published structures (Supplemental Table 2). The zymogen is made up of a 'core' domain (Glu38-Asp313) linked to a C-terminal 'cap' domain (Arg336-Ala474) via the flexible linker, which has weak electron density (Figure 6A).…”

“…This was the first enzyme discovered to be capable of forming peptide bonds posttranslationally within the backbone of protein substrates (Min and Jones, 1994). Interest in exploiting this function for biotechnological applications, on top of efforts to identify similar enzymes capable of peptide backbone transpeptidation, has led to the discovery and engineering of AEPs with varying efficiencies in peptide backbone cleavage, transpeptidation and macrocyclisation, and AEPs with differing preferences for Asp and Asn residues (Nguyen et al, 2014;Yang et al, 2017;Haywood et al, 2018;Harris et al, 2019;James et al, 2019). However, our understanding of the AEP domains critical for determining hydrolase and transpeptidase efficiency is far from complete.…”

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

“…Previous studies showed that the AEP domain in prolegumain is present already in an active conformation ( 21 , 34 ). Zymogenicity resulted solely from the steric blockage of the active site by the AP and LSAM domain.…”

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

“…Multiple reaction monitoring (MRM) was carried out exactly as described previously (94), except trypsin was added to the protein samples in a mass ratio of 1:20. Peptide abundances from each sample were normalised against VDAC in which its abundance was identical between mitochondria from Col-0, dic2-1 and dic2-1/gDIC2 based on western blotting.…”