Different functional role of domain boundaries of Toll-like receptor 4

Toll-like receptors (TLRs) recognize molecules representing danger signals via their ectodomain, while signal transduction is provided by the cytosolic TIR domain that recruits adapter proteins upon dimerization. Since in crystal structures both domains dimerize as rigid bodies, any structural adjustment must be provided by the intermediate segments between the domains. We investigated domain coupling by inserting flexible linkers between the structural domains of TLR4. Insertion of linkers between the transmembrane and cytosolic TIR domain did not affect activation, indicating that TIR domain dimerization is triggered by proximity. In contrast, insertion of a linker between the transmembrane and ectodomain or within the ectodomain decreased activation proportionally with the length of the linker. This suggests the requirement for tight coupling of the ectodomain to the membrane, which may facilitate its interaction with ligand, promote dimerization and prevent interaction with the cell-membrane surface. Native linker sizes of TLR4 orthologs support these conclusions.