Structural Basis for Ca²⁺-Dependent Activation of a Plant Metacaspase

Abstract

<jats:title>Abstract</jats:title><jats:p>Plants metacaspases mediate programmed cell death in development <jats:sup>1,2</jats:sup>, biotic and abiotic stresses <jats:sup>3</jats:sup>, damage-induced immune response <jats:sup>4</jats:sup>, and resistance to pathogen attack <jats:sup>5</jats:sup>. Most metacaspases require Ca<jats:sup>2+</jats:sup> for their activation and substrate processing <jats:sup>6–8</jats:sup>. However, the Ca<jats:sup>2+</jats:sup>-dependent activation mechanism remains elusive <jats:sup>9–11</jats:sup>. Here we report the crystal structure of Metacaspase 4 from <jats:italic>Arabidopsis thaliana</jats:italic> (<jats:italic>At</jats:italic>MC4) that modulates Ca<jats:sup>2+</jats:sup>-dependent, damage-induced plant immune defense. The <jats:italic>At</jats:italic>MC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of <jats:italic>At</jats:italic>MC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca<jats:sup>2+</jats:sup>. Our analysis provides insight into the Ca<jats:sup>2+</jats:sup>-dependent activation of <jats:italic>At</jats:italic>MC4 and lays the basis for tuning its activity in response to stresses that may help engineer more sustainable crops for production of food and biofuel.</jats:p>

Plant type I metacaspases are proteolytically active proteases despite their hydrophobic nature

“…1A) [35], and the second from Saccharomyces cerevisiae, ScMCA-I (before Yca1, [36]). Recently, the first structure of an Arabidopsis type II metacaspase, AtMCA-IIa (before AtMC4), was also resolved [15]. However, despite intensive research over the past two decades, no recombinant and proteolytically active proteases from any organism of the green lineage could be produced heterologously in E. coli.…”

“…Regardless of subtype or calcium dependence, all metacaspases exhibit trypsin-like proteolytic activity as they cleave their substrates after the positively charged amino acid residues Arg or Lys [7,9,[11][12][13]. However, in contrast to type II metacaspases, which undergo rapid autoproteolysis after activation [14,15], type I and III metacaspases exhibit limited proteolysis within the p20-p10 and p10-p20 core structures, respectively, and retain their proteolytic activity even upon prolonged incubation in calcium [7,16]. It should be noted, however, that while several type II metacaspases have been successfully recombinantly expressed in bacteria to date [9,14,[17][18][19][20][21][22], only type I metacaspases from yeasts [23,24] or protozoa [25][26][27], but none from any photosynthetic organism, have been heterologously expressed and characterized in vitro, despite intensive research in planta.…”

Apoptotic-like PCD inducing HRC gene when silenced enhances multiple disease resistance in plants

“…The HRC gene, triggered by the CaM, significantly enhanced Ca 2+ concentration in the cytosol. The increased Ca 2+ can regulate downstream R genes, such as metacaspases and endonucleases, to induce AL-PCD based on two mechanisms: (1) Membrane blebbing and breakdown: The plants have metacaspases, cysteine-dependent multifunctional proteins, that are Ca 2+ responsive and they can induce cell proliferation and breakdown of membrane and DNA, the hallmarks of AL-PCD 14,40 . In potato, the metacaspase gene (StMC7) was significantly induced following Phytophthora and Alternaria inoculations, but the expression was significantly reduced when StHRC was silenced.…”

“…The increase in Ca 2+ concentrations in the cytosol and organelles induce the breakdown of plasma membrane and other organelle membranes, resulting in cell shrinkage. The increase in Ca 2+ concentration in the cytosol by HRC, can also trigger Ca 2+ responsive metacaspases, that can induce breakdown of membranes and DNA 14,40 . The metacaspase, StMC7, was highly upregulated in RB with StHRC, following inoculation of P. infestans and A. solani 35 .…”

Structure-function analysis of a calcium-independent metacaspase reveals a novel proteolytic pathway for lateral root emergence

“…To date, two protein crystal structures of type I MCAs are published from Saccharomyces cerevisiae and Trypanosoma brucei (TbMCA-Ib) (McLuskey et al, 2012;Wong et al, 2012), and one type II calcium dependent MCA from Arabidopsis thaliana (AtMCA-IIa/AtMC4) (Zhu et al, 2020). The core of all three structures is a typical caspase/hemoglobinase alpha-beta-alpha sandwich fold (Aravind & Koonin, 2002).…”