Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases

Jackson, Mark A.; Gilding, Edward K.; Shafee, Thomas; Harris, Karen S.; Kaas, Quentin; Poon, Simon K.; Yap, Kuok; Jia, Husen; Guarino, Rosemary F.; Chan, Lai Yue; Durek, Thomas; Anderson, Marilyn A.; Craik, D.J.

doi:10.1038/s41467-018-04669-9

Cited by 119 publications

(182 citation statements)

References 51 publications

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“…One attractive possibility is that residues within LAD1 (the gatekeeper), LAD2 (this work), and MLA cooperate to determine protease vs. ligase activity. In this respect, we note that the presence of a truncated MLA alone (24) does not necessarily imply a ligase activity, because VcAEP, which Fig. 6.…”

Section: Discussionmentioning

confidence: 93%

Structural determinants for peptide-bond formation by asparaginyl ligases

Hemu

Sahili

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

100

269

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Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptideproducing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. peptide ligase | data mining | ligase-activity determinant

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Section: Discussionmentioning

confidence: 93%

Structural determinants for peptide-bond formation by asparaginyl ligases

Hemu

Sahili

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

100

269

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“…In comparison with 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 ., ). 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 ., ). Moreover, using molecular dynamics simulations Zauner et al .…”

Section: Discussionmentioning

confidence: 97%

“…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 ., ). Furthermore, recent sequence analysis and molecular modeling have revealed a range of residues that together contribute to ligation efficiency (Jackson et al ., ; James et al ., ; Zauner et al ., ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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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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Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases

Cited by 119 publications

References 51 publications

Structural determinants for peptide-bond formation by asparaginyl ligases

Structural determinants for peptide-bond formation by asparaginyl ligases

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

Vacuolar processing enzymes in the plant life cycle

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