Structures of gasdermin-D C-terminal domain reveal mechanisms of autoinhibition

Abstract

Abstract Gasdermin-D (GSDMD) is a member of the gasdermin family, and is a critical mediator for host defense against microbial infection and danger signals. Inflammatory caspases (caspases-1, 4, 5, and 11) cleave GSDMD to generate an N-terminal cleavage fragment (GSDMD-N) that oligomerizes in cell membrane to form ring-like pores during pyroptosis, an informatory form of programmed cell death. In addition, the GSDMD membrane pores facilitate the release of inflammatory cytokines such as IL-1b and IL-18. However, how GSDMD-N is inhibited by the C-terminal domain of GSDMD (GSDMD-C) in the rest state is unknown. In addition, the mechanisms for the release of GSDMD-N from GSDMD-C upon caspase cleavage and the formation of membrane pores have not been established. Here we report the crystal structures of the C-terminal domains of human and murine GSDMD protein. Even though the overall structures of the two GSDMD-C structures are similar to that from mGSDMA3, two regions of the helical structures in GSDMD-C are significantly different from that in mGSDMA3. Overexpression of GSDMD-N induces lysis of the bacterial expression host, while concomitant expression of GSDMD-C reduces such bactericidal activities. Mutation of GSDMD-C residues involved in autoinhibition attenuates the intramolecular domain-domain interactions, as well as enhances pyroptosis in 293T cells transiently expressing GSDMD. Our study thus reveals mechanisms of autoinhibition of GSDMD, and suggests potential therapeutic application of GSDMD as a bactericidal agent.