Mechanisms of Gasdermin Recognition by Proteases

Abstract

Members of the gasdermin family contain positively charged N-terminal domains (NTDs) capable of binding phospholipids and assembling membrane pores, and C-terminal domains (CTDs) that bind the NTDs to prevent pore formation in the resting states. The flexible NTD-CTD linker regions of gasdermins are highly variable in length and sequences, which may be attributable to gasdermin recognition by diverse proteases. In addition, protease cleavage within the NTDs is known to inactivate several gasdermin family members. Recognition and cleavage of the gasdermin family members by different proteases share common and distinct features at the protease active sites, as well as exosites recently identified for the inflammatory caspases. Utilization of exosites may strengthen enzyme-substrate interaction, improve efficiency of proteolysis, and enhance substrate selectivity. It remains to be determined if the dual site recognition of gasdermin D (GSDMD) by the inflammatory caspases is employed by other GSDMD-targeting proteases, or is involved in proteolytic processing of other gasdermins. Biochemical and structural approaches will be instrumental in revealing how potential exosites in diverse proteases engage different gasdermin substrates. Different features of gasdermin sequence, structure, expression characteristics, and post-translational modifications may dictate distinct mechanisms of protease-dependent activation or inactivation. Such diverse mechanisms may underlie the divergent physiological and pathological functions of gasdermins, and furnish opportunities for therapeutic targeting of gasdermins in infectious diseases and inflammatory disorders.

Keywords: caspase; cleavage; exosite; gasdermin; protease.

Figure 1.

Exosites from the inflammatory caspases mediate their recognition of GSDMD. Structures of caspase-1 in complex with GSDMD-CTD (A), caspase-4 in complex with GSDMD-CTD (B), caspase-11 in complex with GSDMD-CTD (C), and caspase-1 in complex with the full-length GSDMD (D) are shown in ribbons with the sidechains of the exosite residues displayed. (E) The structure of a dimeric caspase-1 is shown with the active sites, exosites, and allosteric site marked. (F) Sequence alignment adjacent to the active site Cys residues and the exosite residues are shown for different caspases, with the active site Cys in red, and exosite residues from the inflammatory caspases in green and underlined. The equivalent hydrophobic residues from other caspases are in green, and non-hydrophobic resdues are in black.

Figure 2.

Structures of the GSDMD-CTD and GSDMB-CTD reveal different pockets at the actual or potential interface with the inflammatory caspase exosites. (A) The left panel shows the structure of the GSDMD-CTD (gold) bound to the caspase-1 exosite (green). The right panel shows the GSDMD-CTD as a surface presentation with the hydrophobic residues binding the caspase exosite marked in yellow. (B) The left panel shows the equivalent view of the GSDMB-CTD. The right panel marks the equivalent GSDMB residues to those in (A).