Structural and Mechanistic Insights Into GSDME Pore Formation and Functional Regulation

ABSTRACT Gasdermin E (GSDME) is a unique member of the gasdermin family linked to deafness, cancer, and neurodegenerative diseases, and mediates chemotherapy‐induced pyroptosis; however, its pore‐forming mechanism remains poorly understood. Here, we present cryo‐EM structures of GSDME pores with oligomeric stoichiometries ranging from 23 to 29 protomers. Using mutagenesis, liposome leakage assays, and analysis of cancer‐associated mutations, we identified key residues responsible for membrane binding, shallow membrane insertion, and oligomerization through three distinct structural interfaces. Molecular dynamics simulations reveal dynamic lipid recruitment and specificity: Anionic TMCL2 is selectively recruited by basic residues to facilitate shallow membrane penetration, while zwitterionic DPhPC solvates polar protein regions. Within the assembled 25‐mer pore, specific lipids act as structural stabilizers to reinforce the intermolecular hydrogen bond network. Our findings support a stepwise, time‐dependent mechanism underlying GSDME pore assembly. This study provides critical mechanistic insights into GSDME function and may guide the development of targeted therapeutics for cancers and neurodegenerative diseases.