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. 2016 Jan 29;291(5):2271-87.
doi: 10.1074/jbc.M115.672550. Epub 2015 Dec 1.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

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Free PMC article

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

Michael W Risør et al. J Biol Chem. .
Free PMC article

Abstract

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.

Keywords: Venus flytrap; cysteine proteases; digestion; enzyme; enzyme structure; papain; plant biochemistry; plant carnivory; plant physiology; proteinase.

Figures

FIGURE 1.
FIGURE 1.
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 Asn98p 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, Cys26 and His165, 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.
FIGURE 2.
FIGURE 2.
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 mm) 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”).
FIGURE 3.
FIGURE 3.
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 Asn98p 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 mm E-64. Fits to a two-state unfolding model resulted in Tm values of 72.4 ± 0.1, 73.0 ± 0.1, and 42.7 ± 0.1 °C, respectively.
FIGURE 4.
FIGURE 4.
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.
FIGURE 5.
FIGURE 5.
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 P1 site verified by MALDI-MS. Two boldface positions for peptides 8 and 9 indicate identified cleavage after both P1 positions. C, substrate P3–P1′ residue distribution with R/K set to P1 from substrates with R/K in the Zaa or Yaa position. A clear preference for hydrophobic P2 residues (L/I or F/Y) is seen with a minor fraction of N/Q or R/K. Gly at P1′ is part of the linker sequence in the IQFP substrate. D, substrate P3–P1′ residue distribution for peptides with validated cleavage after Gly in the linker sequence (as P1 position). A strong preference for hydrophobic residues at P2 and R/K in P3 is observed. E, substrate P3–P1′ 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 P2 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.
FIGURE 6.
FIGURE 6.
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.
FIGURE 7.
FIGURE 7.
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 Cys26 and occupies the active site cleft and the S1 and S2 subsites (purple). His165 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.
FIGURE 8.
FIGURE 8.
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 Cys26. 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 S2 pocket of dionain-1 result in a spacious cavity due to Ala139 and Thr70. C, surface presentation of the complete substrate binding cleft in dionain-1 with subsite indication and coloring of important charged groups (S1′: Gln148; S1: Cys66, Asn67, and Asp164; S2: Gln217; S3: Asn62, Asp63, and Asn67). 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 (Asp63-Arg64), located in the loop region from Cys57 to Cys66, and results in a protrusion toward the S3-S4 subsites. Loop 2 is 3-residue insert (Ala94-Gly95-Gly96) in the N-terminal domain. Loop 3 is a 6-residue insert (Pro176–Ser181) in the C-terminal domain that expands an antiparallel β-sheet.
FIGURE 9.
FIGURE 9.
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.

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