Gasdermin D cysteine residues synergistically control its palmitoylation-mediated membrane targeting and assembly

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues. Synopsis Gasdermin D (GSDMD) permeabilizes the plasma membrane during pyroptosis. This work reveals a complex process of GSDMD pore assembly involving specific cysteine residues mediating its membrane targeting and oligomerization. GSDMD membrane targeting is promoted by the synergistic action of Cys192 S-palmitoylation and the presence of negatively-charged lipids. At the membrane, GSDMD preassembles into dimeric and trimeric building units from which higher-order oligomers are generated. Dimer and trimer formation is redundantly mediated by either Cys192 or the Cys39/Cys57 pair. Higher-order oligomerization requires the cooperative action of Cys39/Cys57 and Cys192. The cysteine residue pair Cys39/Cys57 and palmitoylation of Cys192 mediate Gasdermin D membrane targeting and oligomer formation.