Mechanism of membrane pore formation by human gasdermin‐D

TLDR

Gasdermin‑D (GSDMD) mediates pyroptosis by cleaving its N‑terminal domain, which inserts into cellular membranes and oligomerizes to permeabilize them, yet the detailed mechanisms of insertion, oligomerization, and pore formation remain poorly understood. This study uses high‑resolution (≤2 nm) atomic force microscopy to delineate how GSDMDNterm inserts into and assembles within membranes. AFM reveals that GSDMDNterm inserts preferentially into membranes enriched in phosphatidylinositol‑4,5‑bisphosphate, is inhibited by cholesterol, and assembles into arc, slit, and ring oligomers that form transmembrane pores, a process independent of the caspase that cleaves GSDMD. Time‑lapse AFM shows arc‑shaped oligomers transforming into larger slit‑shaped and stable ring‑shaped structures, leading to a mechanistic model of pore assembly that is likely conserved across the gasdermin family.

Abstract

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 (GSDMDNterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high-resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm 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 GSDMDNterm 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 GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.