Mechanism of membrane pore formation by human gasdermin‐D

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.

Gasdermins and their role in immunity and inflammation

“…Studies focusing on GSDMD have reported 16 protomer-shaped pores with inner diameters of 12–14 nm (Aglietti et al, 2016; Ding et al, 2016; Liu et al, 2016; Sborgi et al, 2016); however, these studies have not examined the crystal structure of GSDMD. In contrast, studies by Sborgi et al (2016) and Mulvihill et al (2018) showed that GSDMD pores could instead be averaging ∼20 nm, closer to the diameter of the GSDMA3 pore.…”

Single-molecule kinetics of pore assembly by the membrane attack complex

“…Consistent with the description of these two distinct kinetic steps, the vast majority of end-point MACs in our data are complete, ring-shaped pores. If the rate of initial C9 insertion were faster than the subsequent oligomerization reaction, kinetically-trapped, arc-shaped assemblies would occur due to monomer depletion, as has been observed for other pore-forming proteins 27,28,31 . To determine whether the kinetic bottleneck is attributed to C8 incorporation or insertion of the first C9, we allowed an extended temporal window after the addition of C8 and prior to adding C9 (SI Fig.…”

Growing a gasdermin pore in membranes of pyroptotic cells

“…The findings made by Mulvihill et al provide detailed mechanistic insights into transmembrane pore assembly by GSDMD Nterm . Given the conserved pore‐forming activity in the entire gasdermin family, the Mulvihill study (Mulvihill et al , ), together with the recently determined Cryo‐EM structure of GSDMA3 pores (Ruan et al , ), builds an elegant framework for understanding membrane pore formation during other gasdermin‐mediated pyroptosis that are associated with diverse pathophysiological processes.…”

“…family, the Mulvihill study (Mulvihill et al, 2018), together with the recently determined Cryo-EM structure of GSDMA3 pores (Ruan et al, 2018), builds an elegant framework for understanding membrane pore formation during other gasdermin-mediated pyroptosis that are associated with diverse pathophysiological processes.…”