Mechanism of membrane pore formation by human gasdermin-D

Gasdermin-D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N-terminal domain (GSDMD(Nterm)) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMD(Nterm) insertion, oligomerization, and pore formation are poorly understood. Here, we apply high-resolution (2nm) atomic force microscopy (AFM) to describe how GSDMD(Nterm) inserts and assembles in membranes. We observe GSDMD(Nterm) inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMD(Nterm) assembles arc-, slit-, and ring-shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5. Using time-lapse AFM, we monitor how GSDMD(Nterm) assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. Our observations translate into a mechanistic model of GSDMD(Nterm) transmembrane pore assembly, which is likely shared within the gasdermin protein family. Phosphatidylinositide (PI(4,5)P2) increases gasdermin-D pore formation. Cholesterol reduces gasdermin-D pore formation.