Sublytic gasdermin-D pores captured in atomistic molecular simulations

Abstract

Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases activated by the inflammasome, the GSDMD N-terminal domain (GSDMDNT) assembles on the inner leaflet of the plasma membrane and induces the formation of large membrane pores. We use atomistic molecular dynamics simulations to study GSDMDNT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMDNT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS) head-groups and describe differential lipid binding between the pore and prepore conformations. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMDNT oligomers form stable, water-filled and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation by lipid detachment from GSDMDNT arcs and lipid efflux from partial rings. We find that that high-order GSDMDNT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMDNT rings seen in experiment. Our simulations provide a detailed view of key steps in GSDMDNT-induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis. SignificanceGasdermins execute pyroptotic membrane perforation that is responsible for the release of inflammatory signals and ultimately leads to lytic cell death. They assemble into an approximately 20 nm wide transmembrane {beta}-barrel pore across the plasma membrane. With atomistic molecular simulations of gasdermin-D in a realistic asymmetric plasma membrane mimetic, we show that already small oligomers can form stable water-filled and ionconducting pores. Simulations of larger oligomeric assemblies reveal instabilities in the circular prepore and demonstrate pathways to the formation of slit and ring-shaped pores. Our work gives structural and dynamic insight into how small membrane pores emerge that dissipate the ionic gradient of the cell, but not yet cause cell lysis.