Structural basis of ribosomal peptide macrocyclization in plants

Haywood, Joel; Schmidberger, J.W.; James, Amy M.; Nonis, Samuel G.; Sukhoverkov, Kirill V.; Elias, Mikael; Bond, Charles S.; Mylne, Joshua S.

doi:10.7554/elife.32955

Cited by 61 publications

(153 citation statements)

References 84 publications

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“…Dissociation of this cap domain occurs upon a shift to a low pH environment where in trans self-cleavage at a flexible linker region occurs. Although previous attempts at producing recombinant butelase 1 failed (Nguyen et al, 2014), by using an E. coli strain optimized for recombinant proteins with disulfide bonds (Lobstein et al, 2012) we found we could successfully express and purify recombinant butelase 1 as we have done for four other plant AEPs (Bernath-Levin et al, 2015;Haywood et al, 2018). The recombinant protein possessed a 6-His tag at its N-terminus, followed by a Gly-Ser linker and 462 residues of butelase 1 from Ile21 to Val482 (Supplemental Figure 1).…”

Section: Crystal Structure Of Butelasementioning

confidence: 71%

“…Recent structural studies of AEPs have identified subtle amino acid changes around the active site and substrate binding domain that could influence their ability to perform a transpeptidation reaction (Yang et al, 2017;Haywood et al, 2018). The crystal structure of butelase 1 shows that residues surrounding the catalytic His are conserved in asparaginyl endopeptidases (Figure 1C).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Crystal Structure Of Butelasementioning

confidence: 99%

“…The labelling reaction was stopped by the addition of 10 µL of 6x SDS-PAGE loading buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 0.002% Bromophenol Blue, 0.7135 M (5%) β-mercaptoethanol, 10% glycerol). Proteins were separated using 4-12% Bis-Tris SDS-PAGE gels as previously described (Lu et al, 2015;Haywood et al, 2018). Labelled proteins were visualized in gel with excitation and emission wavelengths of 532 and 580 nm respectively, using a Typhoon 9500 (GE Healthcare).…”

Section: Bodipy Imaging Of Butelase 1 and Haaep2mentioning

confidence: 99%

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

James

Haywood

Leroux

et al. 2018

Preprint

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Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. 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 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 either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

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Section: Crystal Structure Of Butelasementioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Section: Crystal Structure Of Butelasementioning

confidence: 99%

Section: Bodipy Imaging Of Butelase 1 and Haaep2mentioning

confidence: 99%

See 2 more Smart Citations

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

James

Haywood

Leroux

et al. 2018

Preprint

Self Cite

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“…During the generation of GPI-anchor protein, a C13 protease, GPI-anchor transamidase, mediates the peptide ligation step (Benghezal et al, 1996;Mottram et al, 2003;Bundy et al, 2016). The peptide ligation activity of VPE is required for the formation of toxic cyclic peptides (cyclotides) in plants (Min & Jones, 1994;Saska et al, 2007;Mylne et al, 2011;Nguyen et al, 2014;Bernath-Levin et al, 2015;Yang et al, 2017;Haywood et al, 2018;Jackson et al, 2018); this aspect of VPE function is highlighted below when discussing the important role of VPE in plant defense against insects. In this review, we discuss the functions of VPE during the plant life cycle and summarize the most recent development in the field.…”

Section: Introductionmentioning

confidence: 99%

Vacuolar processing enzymes in the plant life cycle

et al. 2019

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Summary Vacuolar processing enzyme (VPE) is a cysteine‐type endopeptidase that has a substrate‐specificity for asparagine or aspartic acid residues and cleaves peptide bonds at their carboxyl‐terminal side. Various vacuolar proteins are synthesized as larger proprotein precursors, and VPE is an important initiator of maturation and activation of these proteins. It mediates programmed cell death (PCD) by provoking vacuolar rupture and initiating the proteolytic cascade leading to PCD. Vacuolar processing enzyme also possesses a peptide ligation activity, which is responsible for producing cyclic peptides in several plant species. These unique functions of VPE support developmental and environmental responses in plants. The number of VPE homologues is higher in angiosperm species, indicating that there has been differentiation and specialization of VPE function over the course of evolution. Angiosperm VPEs are separated into two major types: the γ‐type VPEs, which are expressed mainly in vegetative organs, and the β‐type VPEs, whose expression occurs mainly in storage organs; in eudicots, the δ‐type VPEs are further separated within γ‐type VPEs. This review also considers the importance of processing and peptide ligation by VPE in vacuolar protein maturation.

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On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

et al. 2023

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Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed ‘OaAEP1b‐C247A‐∆351’ could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation.

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Structural basis of ribosomal peptide macrocyclization in plants

Cited by 61 publications

References 84 publications

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

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

Vacuolar processing enzymes in the plant life cycle

On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

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