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Javid, M.* ; Dömling, A.* ; Nikkhah, M.* ; Popowicz, G.M. ; Hashemabadi, M.* ; Shahverdi, A.R.* ; Sepehrizadeh, Z.* ; Ebrahim-Habibi, A.* ; Hosseinkhani, S.*

Geometry-dependent interfaces shape NLRP3 pyrin domain assembly.

Int. J. Biol. Macromol. 375:153422 (2026)
DOI PMC
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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Caps-associated Mutations ; Homotypic Pyd Assembly ; Interfacial Geometry ; Molecular Dynamics Simulations ; Nlrp3 Inflammasome ; Protein–protein Interactions ; Pyrin Domain (pyd) ; Structure-guided Modulation
ISSN (print) / ISBN 0141-8130
e-ISSN 1879-0003
Journal International Journal of Biological Macromolecules
Quellenangaben Volume: 375, Issue: , Pages: , Article Number: 153422 Supplement: ,
Publisher Elsevier
Reviewing status Peer reviewed
Institute(s) Institute of Structural Biology (STB)
Grants Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences
Research Council of Tarbiat Modares University
European Commission through the EPIC
VIDEC
ERA Chair grant ACCELERATOR
ERC Advanced Grant AMADEUS
National Institute for Cancer Research-Programme EXCELES
Cancer Research Czech Republic
Dutch Cancer Society (KWF Kankerbestrijding)