Simon Stael, Igor Sabljić, Dominique Audenaert, +18 , and Frank Van Breusegem

PNAS May 22, 2023; 120 (22) e2303480120

Significance

Metacaspases are widely expressed throughout all kingdoms of life, except mammals. A detailed understanding of their protease activity has been hampered by a paucity of structural information and small molecule inhibitors. We solved the protein structure of an atypical plant metacaspase and, from a chemical library of 10,000 compounds, identified an unknown class of small molecule metacaspase inhibitors. These inhibitors disrupted lateral root emergence in plants, effectively overcoming genetic redundancy. The structural data can aid the understanding of activation mechanism for metacaspases and related proteases, including caspases and paracaspases in mammals, and the inhibitors can form the basis for development of tools and drugs to target metacaspases.

Abstract

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure–function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

See https://www.pnas.org/doi/10.1073/pnas.2303480120

Fig. S2. Close-up views of the catalytic centre and AIM (autoinhibitory motif) binding in the S1 and S2’ pockets. (A) Polar interactions/hydrogen bonds between the P1 residue and protein at the catalytic centre and the S1 pocket, Arg183 in AtMCA-IIf (left panel) and Lys225 in AtMCA-IIa (right panel). Interaction distances in Å are shown by numbers, in red for weaker bonds (3.5 Å or longer). The red-dotted circle indicates the oxyanion hole. (B) Binding of the AIM in the S1 and S2’ pockets in the AtMCA-Iif C147A structure. The S1 and S2´subsites are encircled with red-dotted lines. Different regions are color coded as: p20, blue; linker, green; AIM, yellow; UNK, red; p10, yellow. All residues in the AIM motif are shown, in stick representation, as well as selected and pocket-forming residues