Ultrastructural organization and dynamics of TIRAP filaments

TIRAP (MAL) is an essential adaptor protein in Toll-like receptor (TLR) signaling, bridging activated receptors to downstream effectors such as MyD88 to initiate pro-inflammatory responses. Assembly of TLR signaling complexes is driven by homotypic interactions between Toll/interleukin-1 receptor (TIR) domains. Although previous studies demonstrated that isolated TIR domains of TIRAP can form filaments in vitro, the structural organization and membrane-dependent regulation of full-length TIRAP remained poorly understood. Here, we report a 3.3 [A] cryo-electron microscopy (cryo-EM) structure of full-length human TIRAP filaments combined with the first super-resolution imaging of TIRAP assemblies at the plasma membrane of cells. Using complementary lipid-binding assays on supported lipid bilayers (SLBs) and functional live-cell nanopatterning analysis, we identify a critical role of the N-terminal phosphoinositide-binding motif (PBM) in governing filament geometry and assembly dynamics, plasma membrane partitioning, and effector coupling capacity. Collectively, our multimodal analysis provides a comprehensive structure-function framework of TIRAP, highlighting the intricate features of the PBM as a regulator controlling spatiotemporally defined filament assembly and efficient signal induction at the plasma membrane.