. 2016 Jan 29;291(5):2271-87.
Epub 2015 Dec 1.
Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid
Free PMC article
Item in Clipboard
Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid
J Biol Chem
Free PMC article
Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.
Venus flytrap; cysteine proteases; digestion; enzyme; enzyme structure; papain; plant biochemistry; plant carnivory; plant physiology; proteinase.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Pre-pro-dionain-1 sequence and maturation.
A, Venus flytrap digestive fluid was subjected to SDS-PAGE, and the resolved proteins were blotted to a PVDF membrane and visualized by Coomassie Brilliant Blue staining. A major protein band was observed at ∼45 kDa and subjected to N-terminal sequencing. The obtained sequence (DVPAAVD) corresponded to the N-terminal of dionain-1. B, schematic representation of the full-length dionain-1 cysteine protease (pre-pro-dionain-1) translated from the 5′-race PCR-generated cDNA. Sequence features include the signal peptide ( underlined), the pro-domain (residues p1–p100) containing Asn 98p as possible N-linked glycosylation site (*), the cleavage site for automaturation ( arrow), and the mature protease (residues 1–223) with the active site catalytic dyad, Cys 26 and His 165, indicated in boldface red type. C, SDS-PAGE analysis of pro-dionain-1 expressed in P. pastoris. Pro-dionain-1 was extensively glycosylated (WT) but migrated as a single band at ∼50 kDa upon deglycosylation with endo-β- N-acetylglucosaminidase H ( Endo H). *, Endo H band. SDS-PAGE analysis of the purified N98pQ pro-dionain-1 mutant (N98pQ) confirmed the location of the N-linked glycosylation at this position in the pro-domain.
Effect of pH and temperature on pro-dionain-1 automaturation.
A, pro-dionain-1 was incubated at 0.2 mg/ml at the indicated pH values at 37 °C for 45 min. E-64 (1 m m) was added, and the samples were analyzed by SDS-PAGE. Low pH triggered automaturation to produce mature dionain-1 (∼45 kDa, arrow) and degradation of its own N-terminal pro-domain. B, the enzymatic activity was followed by Z-FR-AMC hydrolysis over time as a function of pH, indicated as RFU. Each curve represents triplicate mean values with experimental deviation of <2.5%. The time to reach the maximal enzyme activity (rate plateau) was extracted through first derivative plots (see inset). The curve profiles clearly demonstrated the pH dependence of the automaturation process. C, the maximum enzyme activity (rate plateau value in RFU/s) was plotted as a function of pH. Error bars, plateau value fit error to the triplicate mean derivative plot. The inset illustrates the derivative plot at pH 3.8 and the fitting curve to the automaturation function (see “Experimental Procedures”).
The role of pro-domain glycosylation on automaturation and stability.
A, SDS-PAGE analysis of WT and deglycosylated pro-dionain-1 after indicated incubation times at pH 4.0 revealed that automaturation proceeded to a similar extent, independent of Asn 98p glycosylation. *, endo-β- N-acetylglucosaminidase H band. B, thermal denaturation profiles of WT, deglycosylated, and N98pQ pro-dionain-1 measured by CD in the presence of 2 m m E-64. Fits to a two-state unfolding model resulted in T values of 72.4 ± 0.1, 73.0 ± 0.1, and 42.7 ± 0.1 °C, respectively. m
Effect of pH and temperature on dionain-1 activity.
A, normalized dionain-1 and papain product conversion rate of Z-FR-AMC as a function of pH. Dionain-1 displays a skewed bell-shaped curve with pH optimum around pH 6. B, normalized end point product conversion after 15-min incubation at the indicated temperatures. Error bars, triplicate S.D.
Substrate profiling of dionain-1 using IQFPs.
A, intensity-weighted score distribution of residue types in positions Xaa, Yaa, and Zaa found in cleaved substrates above a threshold of 1000 RFU (in total 168 motifs). The analysis demonstrated a preference for residues L/I and R/K. B, top 10 ranked substrate pools listed by intensity score. Boldface positions indicate the P 1 site verified by MALDI-MS. Two boldface positions for peptides 8 and 9 indicate identified cleavage after both P 1 positions. C, substrate P 3–P 1′ residue distribution with R/K set to P 1 from substrates with R/K in the Zaa or Yaa position. A clear preference for hydrophobic P 2 residues (L/I or F/Y) is seen with a minor fraction of N/Q or R/K. Gly at P 1′ is part of the linker sequence in the IQFP substrate. D, substrate P 3–P 1′ residue distribution for peptides with validated cleavage after Gly in the linker sequence (as P 1 position). A strong preference for hydrophobic residues at P 2 and R/K in P 3 is observed. E, substrate P 3–P 1′ residue distribution (weighted residue frequencies) of the observed β-casein peptide cuts after overnight in-gel digest by dionain-1. Peptide identification was done by LC-MS/MS. Also, cleavage with P 2 His (1 of 2 possible), Trp (1 of 1), and Met (1 of 4 possible) was observed but is excluded from the pie diagram shown here. F, relative activities of dionain-1 and papain against different di- and tri-peptide AMC substrates, normalized to the rate of Z-FR-AMC hydrolysis.
Dionain-1 digest of
Drosophila proteins. A, extracts of Drosophila (three animals) were treated with increasing amounts of dionain-1 (0, 0.625, 1.25, 5, and 10 μg). The samples were analyzed by SDS-PAGE ( lanes 1–6), including a control incubated without fly extract ( lane 7). Dionain-1 treatment resulted in a general reduction in protein content. The bands selected for MS analysis all contained myosin heavy chain or fragments thereof. The most susceptible bands to dionain-1 treatment are marked with asterisks. B, the dionain-1-treated extracts were analyzed by LC-MS/MS, and the relative protein amount was determined by the exponentially modified protein abundance index protocol. The six most abundant proteins are displayed and account for more than 50% of total protein mass. In agreement with the SDS-PAGE analysis, myosin heavy chain is highly prone to degradation by dionain-1. Error bars, triplicate S.D.
The crystal structure of dionain-1.
A, dionain-1 is a classical papain-like cysteine protease with an N-terminal α-helical lobe ( blue), a C-terminal β-sheet lobe ( gray), and three disulfide bridges ( red). E-64 is found covalently linked to Cys 26 and occupies the active site cleft and the S 1 and S 2 subsites ( purple). His 165 of the catalytic dyad is colored in orange. B, B-factor representation of the final model shows only structural ambiguity in a few loop regions. C, superposition of the Cα traces of papain ( pink; PDB entry 1PPP), Cys-EP ( green; PDB entry 1S4V), and dionain-1 ( blue) shows a high degree of similarity and conservation of the overall structural fold.
Structural details of dionain-1. The N-terminal part is shown in blue, and the C-terminal part is shown in gray. A, active site configuration of dionain-1 in complex with E-64 covalently linked to Cys 26. Relevant hydrogen bonds are shown in orange. The initial Fourier map for E-64 is superimposed with the final refined coordinates for the part of E-64 that was modeled. B, the defining residues of the S 2 pocket of dionain-1 result in a spacious cavity due to Ala 139 and Thr 70. C, surface presentation of the complete substrate binding cleft in dionain-1 with subsite indication and coloring of important charged groups (S 1′: Gln 148; S 1: Cys 66, Asn 67, and Asp 164; S 2: Gln 217; S 3: Asn 62, Asp 63, and Asn 67). D, comparison of the active site cleft ( front view) in papain ( purple), Cys-EP ( green), and dionain-1 ( blue). E, location of loop regions with sequence inserts unique to dionain-1 (compared with papain). Loop 1 is a 2-residue insert (Asp 63-Arg 64), located in the loop region from Cys 57 to Cys 66, and results in a protrusion toward the S 3-S 4 subsites. Loop 2 is 3-residue insert (Ala 94-Gly 95-Gly 96) in the N-terminal domain. Loop 3 is a 6-residue insert (Pro 176–Ser 181) in the C-terminal domain that expands an antiparallel β-sheet.
Features of the pro-dionain-1 structure from homologous modeling.
A, sequence alignment of pro-dionain-1, pro-papain, and pro-caricain reveals highly conserved sequence motifs, including ERFNIN ( boxed orange), salt bridges (indicated by squares), and hydrophobic packing motifs (indicated by spheres). Most divergence is seen in the pro-peptide region blocking the active site (subsite filling) and the sequence that follows. Sequence numbers indicate pro-dionain-1 numbering. B, pro-enzyme structures of thermostable pro-papain ( left, PDB entry 3TNX) and pro-caricain ( right, PDB entry 1PCI). Pro-dionain-1 ( middle) was modeled using Phyre2, and the core enzyme was replaced with the crystal structure determined in this study. C, display of the subsite filling by the pro-peptides of the cognate enzymes. Hydrophobic patch residues are colored green, and active site cysteine and histidine are presented by orange and purple surfaces, respectively (pro-caricain carries the H164A mutation). For pro-dionain-1, subsite carbonyl groups in the core enzyme are represented by red surface colors. For pro-papain and pro-caricain, selected core enzyme-stabilizing carbonyl groups are represented by maroon surface colors.
All figures (9)
A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap).
Biosci Biotechnol Biochem. 2011;75(2):346-8. doi: 10.1271/bbb.100546. Epub 2011 Feb 7.
Biosci Biotechnol Biochem. 2011.
Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.
Biochim Biophys Acta. 2014 Feb;1844(2):374-83. doi: 10.1016/j.bbapap.2013.11.009. Epub 2013 Nov 22.
Biochim Biophys Acta. 2014.
A novel insight into the cost-benefit model for the evolution of botanical carnivory.
Ann Bot. 2015 Jun;115(7):1075-92. doi: 10.1093/aob/mcv050. Epub 2015 May 6.
Ann Bot. 2015.
25948113 Free PMC article.
Discovery of digestive enzymes in carnivorous plants with focus on proteases.
PeerJ. 2018 Jun 5;6:e4914. doi: 10.7717/peerj.4914. eCollection 2018.
29888132 Free PMC article.
Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome.
Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4435-E4441. doi: 10.1073/pnas.1702072114. Epub 2017 May 15.
Proc Natl Acad Sci U S A. 2017.
28507139 Free PMC article.
Structure prediction and network analysis of chitinases from the Cape sundew, Drosera capensis.
Biochim Biophys Acta Gen Subj. 2017 Mar;1861(3):636-643. doi: 10.1016/j.bbagen.2016.12.007. Epub 2016 Dec 28.
Biochim Biophys Acta Gen Subj. 2017.
28040565 Free PMC article.
Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis.
Comput Struct Biotechnol J. 2016 Jun 14;14:271-82. doi: 10.1016/j.csbj.2016.05.003. eCollection 2016.
Comput Struct Biotechnol J. 2016.
27471585 Free PMC article.
Research Support, Non-U.S. Gov't
Cysteine Endopeptidases / chemistry*
Cysteine Proteases / chemistry*
Droseraceae / enzymology*
Hydrophobic and Hydrophilic Interactions
Leucine / analogs & derivatives
Plant Proteins / chemistry*
Protein Structure, Tertiary
LinkOut - more resources
Full Text Sources Miscellaneous