Structural determinants for pH-dependent activation of a plant metacaspase

Abstract

Arabidopsis thaliana metacaspase 9 (AtMC9) plays roles in clearing dead cells, forming xylem vessels, and regulating immunity and programmed cell death in plants. The protease's activation is controlled by pH levels, but the exact structural mechanism behind this has not been elucidated. In this work, we report high-resolution crystal structures for AtMC9 under both active (pH 5.5 and pH 4.2) and inactive (pH 7.5) conditions. The three structures are similar except for local conformations where their hydrogen bonding interactions with solvents are mediated through the protonation of specific titratable amino acid residues' side chains. By combining structural analysis, molecular dynamics simulations under constant pHs, and biochemical assays coupled with site-directed mutagenesis, we show that the regulation of AtMC9 activation involves multiple titratable glutamate and histidine residues across the three domains of p20, linker, and p10. Specifically, deprotonated Glu112, His193, and His208 can suppress AtMC9 proteolytic activity, while protonation of Glu255 and His307 at acidic pH may promote it. This study provides valuable insights into the pH-dependent activation of AtMC9 and could potentially lead to improving crops with enhanced immunity and controlled cell death, ultimately increasing agricultural productivity.

Fig. 1. Structure and activity of AtMC9.

a Schematics of major fragments produced in self-cleavage of AtMC9 and its cleavage of the GST-fused substrate PROPEP1 (residues 40-92) (GST-PROPEP1). Created in BioRender. Liu, H. (2025) https://BioRender.com/x35j923. b pH-dependent self-cleavage and activation of AtMC9. Data are representative of at least three independent experiments. c pH-dependent processing of GST-PROPEP1 by AtMC9. Data are representative of at least three independent experiments. d AtMC9 C147G mutant structures at pH 4.2 (gray), pH 5.5 (purple), and pH 7.5 (colored differently by three domains). e Molecular dynamics trajectories showing the distance between the catalytic C147 sulfur and Arg183’s carbonyl carbon over 50 ns at pH 7.5 (green), 5.5 (red), and 4.2 (blue). Source data are provided as a Source Data file.

Fig. 2. A conserved negatively charged residue, Glu112, in the p20 domain contributes to AtMC9 activation and substrate processing.

a Comparison of the interactions involving Glu112 at three different pHs. b Comparison of representative frames of the pH 4.2 (gray), 5.5 (purple), and 7.5 (green/marine/orange) simulations showing interactions involving Glu112. Interactions at each pH are displayed as each pH’s respective color but with pH 7.5 displayed as red. c Self-cleavage in E112K. For the wild-type (WT) sample, pH 8 was used in the incubation to compare with the mutant. Data are representative of two independent experiments. d Substrate GST-PROPEP1 processing by E112K. Data are representative of two independent experiments. Source data are provided as a Source Data file.

Fig. 3. Two histidine residues in the linker domain regulate self-cleavage activation and substrate processing.

a Comparison of the interactions involving H193 and H208 at pH 4.2, 5.5 and 7.5. Interactions and water molecules at each pH are displayed as each pH’s respective color but with pH 7.5 displayed as red. b Comparison of representative frames of the pH 4.2 (gray), 5.5 (purple), and 7.5 (marine/green) simulations showing interactions found in and around the linker domain. c Self-cleavage in H193A. d Self-cleavage in H208A. e Substrate processing of GST-PROPEP1 by H193A. f Substrate processing of GST-PROPEP1 by H208A. WT: For the wild-type AtMC9 sample in panels c and d, pH 8 was used in the incubation to compare with the mutant. Data are representative of two independent experiments for (c–f). Source data are provided as a Source Data file.

Fig. 4. Residues Glu255 and His307 in the p10 domain regulate the self-cleavage activation and substrate processing.

a Interactions involving Glu255 and His307 at pH 4.2, 5.5, and 7.5. Interactions and water at each pH are displayed as each pH’s respective color, but with pH 7.5 displayed as red. b Comparison of representative frames of the pH 4.2 (gray), 5.5 (purple), and 7.5 (marine/orange) simulations showing interactions involving Glu255 and His307. c Self-cleavage in E255A. d Self-cleavage in H309A. e Substrate processing of GST-PROPEP1 by E255A. f Substrate processing of GST-PROPEP1 by H309A. WT: For the wild-type AtMC9 sample in (c, d), pH 8 was used in the incubation to compare with the mutant. Data are representative of two independent experiments for (c–f). Source data are provided as a Source Data file.

Fig. 5. Functional characterization of AtMC9 and its mutants.

The GRRase activity is shown as relative fluorescence units (RFUs) for 60 min with 1-min intervals for H193A (black). E112K (pink), H208A (green), WT (orange), H307A (blue), E255A (purple), and C147G (red) under indicated pHs for 60 min with 1-min intervals. Data are presented as mean ± standard deviation (SD). Error bars represent SD (n = 3 independent technical replicates). a 3.6; b 4.6; c 5.6; d 6.6; e 7.6; f 8.6; g 9.6. h The RFU range for AtMC9 and its mutants at 7 different pHs at the 20 min time point: +++++, >40,000; ++++, >20,000; +++, >10,000; ++, >5000; +, >1000; ±, >200; −, 0. Activities lower than WT are highlighted in blue, and those higher in salmon. Source data are provided as a Source Data file.

Fig. 6. Proposed mechanism of AtMC9 activation and regulation.

Surface model of the structures surrounding the active site region of AtMC9, with the key modulating residues outlined and labeled. Blue down-arrows signify decreased activation. The red up-arrow indicates increased activation. Black spindles represent suppressive functions as Glu112 keeps Cys147 away from cleaving the inhibitory Arg183 in the active site at a neutral pH or higher. In contrast, the Black arrow indicates the activating function of Glu255 and His307, as these residues may push Cys147 toward Arg183 under acidic pH conditions.