Structural basis for GSDMB pore formation and its targeting by IpaH7.8

Abstract

Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis1,2. Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential3–7. Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity4. Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB4. Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB–IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity.

Alternative splicing of GSDMB modulates killer lymphocyte–triggered pyroptosis

“…The sequences of the pore-forming GSDMB4-NT and long form of GSDMB3-NT (3L; NT244) are identical in this region, whereas GSDMB5 has a four-residue insertion, potentially disrupting the belt and shifting residues within the belt (Fig. 2A), in line with a recent cryo-EM analysis of GSDMB5 (30). Structural modeling of GSDMB-NT isoforms indicated that the belt is unstable in isoforms 1, 2, 3S (NT229), and 5, which either lack the β9-β11 hairpin, have the C terminus hanging far from the α3-β6-β9 cavity because of the lack of SerPhe, or both (Fig.…”

“…2F). It is possible that there are subtle lipid binding differences under the detection limit of our assay, because triple charge-reversal mutations of R225-K227-K229 modestly lowered the activity of GSDMB3 in liposomebased experiments (30). Nonetheless, less-aggressive double mutations of R225-K227 to alanines did not affect GSDMB3 activity (31), supporting our postulation that the belt promotes pore formation mainly by an alternative mechanism.…”

“…Therefore, the belt is not part of the disordered linker between the NT and CT domains of GSDMs but rather is an ordered, integral structure within the active NT domain. Cryo-EM structures of the membrane-inserted GSDMB (30,31) published during the revision of this manuscript confirmed the orderedness of the belt.…”

“…2D). Consistently, crystal structures of GSDMB5 showed that the belt is disordered in this isoform (30)(31)(32). Therefore, the belt motif in isoforms 3L and 4, which resembles the stabilizing structures formed in GSDMD and GSDMA3, is necessary for pore formation and unstable in the noncytotoxic isoforms.…”

Regulating GSDMB pore formation: to ignite or inhibit?

“…Recently, several studies have been published to answer the questions of whether GSDMB can induce pyroptosis, how Shigella IpaH7.8 targets GSDMB, and particularly, why IpaH7.8 ubiquitinates human but not mouse GSDMD [10][11][12][13]. In the latest issue of Nature, Wang et al and Zhong et al determined the structures of GSDMB-IpaH7.8 complex using cyrogenic electron microscopy (cryo-EM) and X-ray crystallography, respectively [10,11]. Both structures contain a full-length GSDMB adopting the autoinhibited confirmation and an IpaH7.8 LRR domain interacting with the GSDMB N-terminal pore-forming domain (GSDMB-N).…”