Structural and Mechanistic Insights Into GSDME Pore Formation and Functional Regulation.

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.