Crystal structure of the human NLRP9 pyrin domain suggests a distinct mode of inflammasome assembly

Abstract

Inflammasomes are cytosolic multimeric signaling complexes of the innate immune system that induce activation of caspases. The NOD-like receptor NLRP9 recruits the adaptor protein ASC to form an ASC-dependent inflammasome to limit rotaviral replication in intestinal epithelial cells, but only little is known about the molecular mechanisms regulating and driving its assembly. Here, we present the crystal structure of the human NLRP9 pyrin domain (PYD). We show that NLRP9 PYD is not able to self-polymerize nor to nucleate ASC specks in HEK293T cells. A comparison with filament-forming PYDs revealed that NLRP9 PYD adopts a conformation compatible with filament formation, but several charge inversions of interfacing residues might cause repulsive effects that prohibit self-oligomerization. These results propose that inflammasome assembly of NLRP9 might differ largely from what we know of other inflammasomes.

NLRP9 in innate immunity and inflammation

“…The overall structure of hNLRP9 PYD assumes an antiparallel six‐helical bundle fold 31,32 that is typical for the death domain superfamily that PYD belongs to 33 . Surprisingly, hNLRP9 PYD exited as monomers instead of a higher oligomeric filament structure in solution 31,32 . Consistently, hNLRP9 PYD did not self‐polymerize or promote speck formation of ASC when overexpressed in HEK293 T cells 31 .…”

“…Surprisingly, hNLRP9 PYD exited as monomers instead of a higher oligomeric filament structure in solution 31,32 . Consistently, hNLRP9 PYD did not self‐polymerize or promote speck formation of ASC when overexpressed in HEK293 T cells 31 . This is in great contrast to PYDs in other inflammasome sensors including NLRP3, NLRP6 and AIM2, all of which could oligomerize to act as the nucleation seed for ASC filament assembly 22,34‐36 .…”

“…As the common module shared by all NLR proteins, PYD is not only indispensable for the interaction between NLRs and ASC, but is also crucial for signal amplification of inflammasome pathways through its nucleation‐driven polymerization. The overall structure of hNLRP9 PYD assumes an antiparallel six‐helical bundle fold 31,32 that is typical for the death domain superfamily that PYD belongs to 33 . Surprisingly, hNLRP9 PYD exited as monomers instead of a higher oligomeric filament structure in solution 31,32 .…”

“…Two recent studies 31,32 have solved the crystal structure of PYD derived from human NLRP9 (hNLRP9 PYD ), highlighting the similarities and differences of NLRP9 with other NLRs. As the common module shared by all NLR proteins, PYD is not only indispensable for the interaction between NLRs and ASC, but is also crucial for signal amplification of inflammasome pathways through its nucleation‐driven polymerization.…”

ATP-Binding and Hydrolysis in Inflammasome Activation

“…Moreover, 3D modeling of the NACHT domain suggests stable coordination of ADP and ATP in the binding pocket of NLRP9, although comparatively fewer H-bonds were identified with molecular dynamic simulations using a manually-docked complex [ 47 ]. Finally, structures of the PYD from the human NLRP9 gene reveal a novel orientation for the domain [ 146 , 147 ] which may reflect differences in oligomerization and ATP-dependent inflammasome formation.…”