Geometry-Dependent Interfaces Shape Nlrp3 Pyrin Domain Assembly Publisher Pubmed



Javid M ; Domling A ; Nikkhah M ; Popowicz G ; Hashemabadi M ; Shahverdi A R ; Sepehrizadeh Z ; Ebrahim Habibi A ; Hosseinkhani S
Source: International Journal of Biological Macromolecules Published:2026

Abstract

NLRP3 inflammasome activation requires precise regulation of pyrin domain (PYD)-mediated protein–protein interactions to prevent spontaneous self-association while enabling rapid ASC adaptor recruitment. However, how local PYD interfaces encode the balance between restrained and assembly-competent states remains unclear. Here, we combine structural analysis, atomistic molecular dynamics simulations, targeted mutagenesis, heterotypic native/mutant split-luciferase complementation, ASC recruitment assays, microscale thermophoresis, and mass photometry to define geometry-dependent regulation of NLRP3 PYD self-association. A key feature of this work is the use of heterotypic native/mutant PYD pairings, enabling systematic comparison of interfacial, peripheral, and dual mutations and revealing interaction modes not accessible in homogeneous wild-type or mutant systems.We identify two distinct PYD–PYD geometries: a Type A-like restrained, non-filamentous dimeric contact and a Type B-like filament-compatible geometry that preserves the canonical Ia–Ib interface observed in active inflammasome assemblies. Both retain the six-helix PYD fold, indicating that functional differences arise primarily from interfacial organization rather than global structural changes. Residue-level perturbations further show that regulatory and CAPS-associated mutations remodel PYD assembly through distinct mechanisms. The S5D phosphomimetic substitution exhibits position- and orientation-dependent effects, being tolerated in permissive contexts but disrupting filament-compatible organization in sensitive interfaces. D31V decouples adaptor docking from higher-order assembly, whereas D21H reshapes restrained Type A-like interactions while preserving assembly competence.Together, these findings establish NLRP3 PYD self-association as a geometry-dependent process governed by electrostatic, hydrogen-bonding, and hydrophobic networks. This framework provides structural insight into regulatory modifications and CAPS-associated mutations and highlights PYD interfaces as potential targets for structure-guided modulation of NLRP3 inflammasome signaling. © 2026 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.